Human EPO mimetic hinge core mimetibodies, compositions, methods and uses

ABSTRACT

The present invention relates to at least one novel human EPO mimetic hinge core mimetibody or specified portion or variant, including isolated nucleic acids that encode at least one EPO mimetic hinge core mimetibody or specified portion or variant, EPO mimetic hinge core mimetibody or specified portion or variants, vectors, host cells, transgenic animals or plants, and methods of making and using thereof, including therapeutic compositions, methods and devices.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to mammalian EPO mimetic hinge coremimetibodies, specified portions and variants specific for bologicallyactive proteins, fragment or ligands, EPO mimetic hinge core mimetibodyencoding and complementary nucleic acids, host cells, and methods ofmaking and using thereof, including therapeutic formulations,administration and devices.

2. Related Art

Recombinant proteins are an emerging class of therapeutic agents. Suchrecombinant therapeutics have engendered advances in protein formulationand chemical modification. Such modifications can potentially enhancethe therapeutic utility of therapeutic proteins, such as by increaseinghalf lives (e.g., by blocking their exposure to proteolytic enzymes),enhancing biological activity, or reducing unwanted side effects. Onesuch modification is the use of immunoglobulin fragments fused toreceptor proteins, such as enteracept. Therapeutic proteins have alsobeen constructed using the Fc domain to attempt to provide a longerhalf-life or to incorporate functions such as Fc receptor binding,protein A binding, and complement fixation. One specific and vital roleof the mammalian hematopoietic system is the production of erythrocytes,or red blood cells, which transport oxygen to the various tissues of theanimal's body. The process of producing erythrocytes (“erythropoiesis”)occurs continuously throughout an animal's life span to offseterythrocyte destruction. The typical red blood cell has a relativelyshort life-span, usually 100 to 120 days. Erythropoiesis is a preciselycontrolled physiological mechanism whereby sufficient numbers oferythrocytes are produced to enable proper tissue oxygenation, but notso many as to impede circulation.

Erythropoiesis is now known to be primarily controlled by thepolypeptide erythropoietin (EPO), an acidic glycoprotein. Erythropoietinis produced as the result of the expression of a single copy genelocated in a chromosome of a mammal. The amino acid sequence forrecombinant human EPO (“rHuEPO”) is substantially identical to the aminoacid sequence for EPO obtained from human urinary sources. However, theglycosylation of rHuEPO differs from that of urinary EPO and human serumEPO.

In a healthy mammal, EPO is present in the blood plasma in very lowconcentrations, as the tissues are being sufficiently oxygenated by theexisting number of circulating erythrocytes. The EPO present stimulatesthe production of new erythrocytes to replace those lost to the agingprocess. Additionally, EPO production is stimulated under conditions ofhypoxia, wherein the oxygen supply to the body's tissues is reducedbelow normal physiological levels despite adequate perfusion of thetissue by blood. Hypoxia may be caused by hemorrhaging,radiation-induced erythrocyte destruction, various anemias, highaltitude, or long periods of unconsciousness. In contrast, should thenumber of red blood cells in circulation exceed what is needed fornormal tissue oxygenation, EPO production is reduced.

However, certain disease states involve abnormal erythropoiesis.Recombinant human EPO (rHuEPO) is being used therapeutically in a numberof countries. In the United States, the U.S. Food and DrugAdministration (FDA) has approved rHuEPO's use in treating anemiaassociated with end-stage renal disease. Patients undergoinghemodialysis to treat this disorder typically suffer severe anemia,caused by the rupture and premature death of erythrocytes as a result ofthe dialysis treatment. EPO is also useful in the treatment of othertypes of anemia. For instance, chemotherapy-induced anemia, anemiaassociated with myelodysplasia, those associated with various congenitaldisorders, AIDS-related anemia, and prematurity-associated anemia, maybe treated with EPO. Additionally, EPO may play a role in other areas,such as helping to more quickly restore a normal hematocrit in bonemarrow transplantation patients, in patients preparing for autologousblood transfusions, and in patients suffering from iron overloaddisorders.

Erythropoietin (EPO) is a glycoprotein hormone composed of 165 aminoacids and four carbohydrate chains that functions as the primaryregulator of erythropoiesis by binding to a specific receptor on thesurface of erythrocyte precursor cells. This binding signals theirproliferation and differentiation into mature red blood cells. Theerythropoietin receptor is a 484-amino acid glycoprotein with highaffinity for erythropoietin. For the erythropoietin receptor,ligand-induced homodimerization may be one of the key event that governsactivation.

Erythropoietin has a relatively short half-life. Intravenouslyadministered erythropoietin is eliminated at a rate consistent withfirst order kinetics with a circulating half-life ranging fromapproximately 3 to 4 hours in patients with CRF. Within the therapeuticdose range, detectable levels of plasma erythropoietin are maintainedfor at least 24 hours. After subcutaneous administration oferythropoietin, peak serum levels are achieved within 5-24 hours anddecline slowly thereafter.

Small peptidomimetics of erythropoietin were identified by severalgroups through screening of random phage display peptide libraries foraffinity to the erythropoietin receptor. These sequences have nohomology with erythropoietin. In functional assays several of thesepeptides showed activity, but only 1/100,000^(th) that of recombinanterythropoietin. Although several attempts have been made to increase thepotency of these peptides by preparing covalent dimers or multimers ofpeptidomimetics, these compounds are still 1,000-10,000 fold less activethan erythropoietin on a molar basis and have very short half lives thathas made them not suitable for use as therapeutics.

Accordingly, there is a need to provide improved and/or modifiedversions of EPO therapeutic proteins, which overcome one more of theseand other problems known in the art.

SUMMARY OF THE INVENTION

The present invention provides human EPO mimetic hinge coremimetibodies, including modified immunoglobulins, cleavage products andother specified portions and variants thereof, as well as EPO mimetichinge core mimetibody compositions, encoding or complementary nucleicacids, vectors, host cells, compositions, formulations, devices,transgenic animals, transgenic plants, and methods of making and usingthereof, as described and/or enabled herein, in combination with what isknown in the art.

The present invention also provides at least one isolated EPO mimetichinge core mimetibody or specified portion or variant as describedherein and/or as known in the art. The EPO mimetic hinge core mimetibodycan optionally comprise at least one CH3 region directly linked with atleast one CH2 region directly linked with at least one portion of atleast one hinge region or fragment thereof (H), directly linked with anoptional linker sequence (L), directly linked to at least one EPOmimetic therapeutic peptide (P), optionally further directly linked withat least a portion of at least one variable antibody sequence (V). In apreferred embodiment a pair of a CH3-CH2-hinge-linker-therapeuticpeptide with an optional N-terminal antibody sequence, the pairoptionally linked by association or covalent linkage, such as, but notlimited to, at least one Cys-Cys disulfide bond or at least one CH4 orother immunglobulin sequence. In one embodiment, an EPO mimetic hingecore mimetibody comprises formula (I):((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s),where V is at least one portion of an N-terminus of an immunoglobulinvariable region, P is at least one bioactive EPO mimetic polypeptide, Lis at least one linker sequence, H is least one portion of a nimmunoglobulin variable region, CH2 is at least a portion of animmunoglobulin CH2 constant region, CH3 is at least a portion of animmunoglobulin CH3 constant region, m, n, o p, q, r, and s can beindependently an integer between 0, 1 or 2 and 10, mimicing differenttypes of immunoglobulin molecules, e.g., but not limited to IgG1, IgG2,IgG3, IgG4, IgA1, IgA2, IgM, IgD, IgE, or any subclass thereof, and thelike, or any combination thereof.

Thus, an EPO mimetic hinge core mimetibody of the present inventionmimics at least a portion of an antibody or immnuoglobulin structure orfunction with its inherent properties and functions, while providing atherapeutic peptide and its inherent or acquired in vitro, in vivo or insitu properties or activities. The various portions of the antibody andtherapeutic peptide portions of at least one EPO mimetic hinge coremimetibody of the present invention can vary as described herein incombination with what is known in the art.

The present invention provides, in one aspect, isolated nucleic acidmolecules comprising, complementary, having significant identity orhybridizing to, a polynucleotide encoding specific mimetibodies orspecified portions or variants thereof, comprising at least onespecified sequence, domain, portion or variant thereof. The presentinvention further provides recombinant vectors comprising at least oneof said isolated EPO mimetic hinge core mimetibody nucleic acidmolecules, host cells containing such nucleic acids and/or recombinantvectors, as well as methods of making and/or using such EPO mimetichinge core mimetibody nucleic acids, vectors and/or host cells.

At least one EPO mimetic hinge core mimetibody or specified portion orvariant of the invention mimics the binding of the P portion of themimetibody to at least one ligand, or has at least one biologicalactivity of, at least one protein, subunit, fragment, portion or anycombination thereof.

The present invention also provides at least one isolated EPO mimetichinge core mimetibody or specified portion or variant as describedherein and/or as known in the art, wherein the EPO mimetic hinge coremimetibody or specified portion or variant has at least one activity,such as, but not limited to known biological activities of at least onebioactive peptide or polypeptide corresponding to the P portion ofFormula I. An EPO mimetic hinge core mimetibody can thus be screened fora corresponding activity according to known methods, such as at leastone neutralizing activity towards a protein or fragment thereof.

The present invention also provides at least one composition comprising(a) at least one isolated EPO mimetic hinge core mimetibody or specifiedportion or variant encoding nucleic acid and/or EPO mimetic hinge coremimetibody as described herein; and (b) a suitable carrier or diluent.The carrier or diluent can optionally be pharmaceutically acceptable,according to known methods. The composition can optionally furthercomprise at least one further compound, protein or composition.

The present invention also provides at least one method for expressingat least one EPO mimetic hinge core mimetibody or specified portion orvariant in a host cell, comprising culturing a host cell as describedherein and/or as known in the art under conditions wherein at least oneEPO mimetic hinge core mimetibody or specified portion or variant isexpressed in detectable and/or recoverable amounts.

The present invention further provides at least one EPO mimetic hingecore mimetibody, specified portion or variant in a method orcomposition, when administered in a therapeutically effective amount,for modulation, for treating or reducing the symptoms of at least one ofa bone and joint disorder, cardiovascular disoder, a dental or oraldisorder, a dermatologic disorder, an ear, nose or throat disorder, anendocrine or metabolic disorder, a gastrointestinal disorder, agynecologic disorder, a hepatic or biliary disorder, a an obstetricdisorder, a hematologic disorder, an immunologic or allergic disorder,an infectious disease, a musculoskeletal disorder, a oncologic disorder,a neurologic disorder, a nutritrional disorder, an opthalmologicdisorder, a pediatric disorder, a poisoning disorder, a psychiatricdisorder, a renal disorder, a pulmonary disorder, or any other knowndisorder. (See., e.g., The Merck Manual, 17th ed., Merck ResearchLaboratories, Merck and Co., Whitehouse Station, N.J. (1999), entirelyincoporated herein by reference), as needed in many differentconditions, such as but not limited to, prior to, subsequent to, orduring a related disease or treatment condition, as known in the art.

The present invention further provides at least one EPO mimetic hingecore mimetibody, specified portion or variant in a method orcomposition, when administered in a therapeutically effective amount,for modulation, for treating or reducing the symptoms of, at least oneimmune, cardiovascular, infectious, malignant, and/or neurologic diseasein a cell, tissue, organ, animal or patient and/or, as needed in manydifferent conditions, such as but not limited to, prior to, subsequentto, or during a related disease or treatment condition, as known in theart and/or as described herein.

The present invention also provides at least one composition, deviceand/or method of delivery of a therapeutically or prophylacticallyeffective amount of at least one EPO mimetic hinge core mimetibody orspecified portion or variant, according to the present invention.

The present invention further provides at least one anti-idiotypeantibody to at least one EPO mimetic hinge core mimetibody of thepresent invention. The anti-idiotype antibody includes any protein orpeptide containing molecule that comprises at least a portion of animmunoglobulin molecule, such as but not limited to at least onecomplimetarity determing region (CDR) of a heavy or light chain or aligand binding portion thereof, a heavy chain or light chain variableregion, a heavy chain or light chain constant region, a frameworkregion, or any portion thereof, that competitively binds an EPO receptorbinding region of at least one EPO mimetic hinge core mimetibody of thepresent invention. Such idiotype antibodies of the invention can includeor be derived from any mammal, such as but not limited to a human, amouse, a rabbit, a rat, a rodent, a primate, and the like.

The present invention also provides at least one isolated nucleic acidmolecule comprising, complementary, or hybridizing to, a polynucleotideencoding at least one EPO mimetic hinge core mimetibody anti-idiotypeantibody, comprising at least one specified sequence, domain, portion orvariant thereof. The present invention further provides recombinantvectors comprising said EPO mimetic hinge core mimetibody anti-idiotypeantibody encoding nucleic acid molecules, host cells containing suchnucleic acids and/or recombinant vectors, as well as methods of makingand/or using such anti-idiotype antiobody nucleic acids, vectors and/orhost cells.

The present invention also provides at least one method for expressingat least one EPO mimetic hinge core mimetibody, or EPO mimetic hingecore mimetibody anti-idiotype antibody, in a host cell, comprisingculturing a host cell as described herein under conditions wherein atleast one EPO mimetic hinge core mimetibody or anti-idiotype antibody isexpressed in detectable and/or recoverable amounts.

The present invention also provides at least ore composition comprising(a) an isolated EPO mimetic hinge core mimetibody encoding nucleic acidand/or EPO mimetic hinge core mimetibody as described herein; and (b) asuitable carrier or diluent. The carrier or diluent can optionally bepharmaceutically acceptable, according to known carriers or diluents.The composition can optionally further comprise at least one furthercompound, protein or composition.

The present invention further provides at least one EPO mimetic hingecore mimetibody method or composition, for administering atherapeutically effective amount to modulate or treat at least oneprotein related condition in a cell, tissue, organ, animal or patientand/or, prior to, subsequent to, or during a related condition, as knownin the art and/or as described herein.

The present invention also provides at least one composition, deviceand/or method of delivery of a therapeutically or prophylacticallyeffective amount of at least one EPO mimetic hinge core mimetibody,according to the present invention.

The present invention further provides at least one EPO mimetic hingecore mimetibody method or composition, for diagnosing at least one EPOrelated condition in a cell, tissue, organ, animal or patient and/or,prior to, subsequent to, or during a related condition, as known in theart and/or as described herein.

The present invention also provides at least one composition, deviceand/or method of delivery for diagnosing of at least one EPO mimetichinge core mimetibody, according to the present invention.

In one aspect, the present invention provides at least one isolatedhuman EPO mimetic hinge core mimetibody, comprising at least one P(n)region comprising at least a portion of at least one of SEQ ID NOS:1-30,e.g., as presented in Table 1 below, or optionally with one or moresubstitutions, deletions or insertions as described herein or as knownin the art. In other aspect the present invention provides at least oneisolated human EPO mimetic hinge core mimetibody, wherein the EPOmimetic hinge core mimetibody specifically binds at least one epitopecomprising at least 1-3 of at least one ligand or binding region whichligand binds to at least a portion of at least one of SEQ ID NOS:1-30 aspresented in Table 1 below, or optionally with one or moresubstitutions, deletions or insertions as described herein or as knownin the art.

The at least one EPO mimetic hinge core mimetibody can optionallyfurther at least one of: bind protein with an affinity of at least oneselected from at least 10⁻⁹ M, at least 10⁻¹⁰ M, at least 10⁻¹¹ M, or atleast 10⁻¹² M; substantially neutralize at least one activity of atleast one protein or portion thereof. Also provided is an isolatednucleic acid encoding at least one isolated human EPO mimetic hinge coremimetibody; an isolated nucleic acid vector comprising the isolatednucleic acid, and/or a prokaryotic or eukaryotic host cell comprisingthe isolated nucleic acid. The host cell can optionally be at least oneselected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, 653,SP2/0, 293, HeLa, myeloma, or lymphoma cells, or any derivative,immortalized or transformed cell thereof. Also provided is a method forproducing at least one EPO mimetic hinge core mimetibody, comprisingtranslating the EPO mimetic hinge core mimetibody encoding nucleic acidunder conditions in vitro, in vivo or in situ, such that the EPO mimetichinge core mimetibody is expressed in detectable or recoverable amounts.

Also provided is a composition comprising at least one isolated humanEPO mimetic hinge core mimetibody and at least one pharmaceuticallyacceptable carrier or diluent. The composition can optionally furthercomprise an effective amount of at least one compound or proteinselected from at least one of a detectable label or reporter, ananti-infective drug, a cardiovascular (CV) system drug, a centralnervous system (CNS) drug, an autonomic nervous system (ANS) drug, arespiratory tract drug, a gastrointestinal (GI) tract drug, a hormonaldrug, a drug for fluid or electrolyte balance, a hematologic drug, anantineoplactic, an immunomodulation drug, an ophthalmic, otic or nasaldrug, a topical drug, a nutritional drug, a TNF antagonist, anantirheumatic, a muscle relaxant, a narcotic, a non-steroidanti-inflammatory drug (NTHE), an analgesic, an anesthetic, a sedative,a local anethetic, a neuromuscular blocker, an antimicrobial, anantipsoriatic, a corticosteriod, an anabolic steroid, an erythropoietin,an immunization, an immunoglobulin, an immunosuppressive, a growthhormone, a hormone replacement drug, a radiopharmaceutical, anantidepressant, an antipsychotic, a stimulant, an asthma medication, abeta agonist, an inhaled steroid, an epinephrine or analog, a cytokine,or a cytokine antagonist.

The present invention further provides an anti-idiotype antibody orfragment that specifically binds at least one EPO mimetic hinge coremimetibody of the present invention.

Also provided is a method for diagnosing or treating a disease conditionin a cell, tissue, organ or animal, comprising

(a) contacting or administering a composition comprising an effectiveamount of at least one isolated human EPO mimetic hinge core mimetibodyof the invention with, or to, the cell, tissue, organ or animal. Themethod can optionally further comprise using an effective amount of0.001-50 mg/kilogram of the cells, tissue, organ or animal. The methodcan optionally further comprise using the contacting or theadministrating by at least one mode selected from parenteral,subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracelebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.The method can optionally further comprise administering, prior,concurrently or after the (a) contacting or administering, at least onecomposition comprising an effective amount of at least one compound orprotein selected from at least one of a detectable label or reporter, ananti-infective drug, a cardiovascular (CV) system drug, a centralnervous system (CNS) drug, an autonomic nervous system (ANS) drug, arespiratory tract drug, a gastrointestinal (GI) tract drug, a hormonaldrug, a drug for fluid or electrolyte balance, a hematologic drug, anantineoplactic, an immunomodulation drug, an ophthalmic, otic or nasaldrug, a topical drug, a nutritional drug, a TNF antagonist, anantirheumatic, a muscle relaxant, a narcotic, a non-steroidanti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative,a local anethetic, a neuromuscular blocker, an antimicrobial, anantipsoriatic, a corticosteriod, an anabolic steroid, an erythropoietin,an immunization, an immunoglobulin, an immunosuppressive, a growthhormone, a hormone replacement drug, a radiopharmaceutical, anantidepressant, an antipsychotic, a stimulant, an asthma medication, abeta agonist, an inhaled steroid, an epinephrine or analog, a cytokine,or a cytokine antagonist.

Also provided is a medical device, comprising at least one isolatedhuman EPO mimetic hinge core mimetibody of the invention, wherein thedevice is suitable to contacting or administerting the at least one EPOmimetic hinge core mimetibody by at least one mode selected fromparenteral, subcutaneous, intramuscular, intravenous, intrarticular,intrabronchial, intraabdominal, intracapsular, intracartilaginous,intracavitary, intracelial, intracelebellar, intracerebroventricular,intracolic, intracervical, intragastric, intrahepatic, intramyocardial,intraosteal, intrapelvic, intrapericardiac, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine,intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, ortransdermal.

Also provided is an article of manufacture for human pharmaceutical ordiagnostic use, comprising packaging material and a container comprisinga solution or a lyophilized form of at least one isolated human EPOmimetic hinge core mimetibody of the present invention. The article ofmanufacture can optionally comprise having the container as a componentof a parenteral, subcutaneous, intramuscular, intravenous,intrarticular, intrabronchial, intraabdominal, intracapsular,intracartilaginous, intracavitary, intracelial, intracelebellar,intracerebroventricular, intracolic, intracervical, intragastric,intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, or transdermal deliverydevice or system.

Also provided is a method for producing at least one isolated human EPOmimetic hinge core mimetibody of the present invention, comprisingproviding a host cell or transgenic animal or transgenic plant or plantcell capable of expressing in recoverable amounts the EPO mimetic hingecore mimetibody. Further provided in the present invention is at leastone EPO mimetic hinge core mimetibody produced by the above method.

The present invention also provides at least one method for expressingat least one EPO mimetic hinge core mimetibody, or anti-idiotypeantibody, in a host cell, comprising culturing a host cell as describedherein under conditions wherein at least one EPO mimetic hinge coremimetibody is expressed in detectable and/or recoverable amounts.

The present invention further provides any invention described herein.

DESCRIPTION OF THE FIGURES

FIGS. 1-42 (SEQ ID NOS:31-72) show examples of heavy/light chainvariable/constant region sequences, frameworks/subdomains andsubstitutions, portions of which can be used in Ig derived proteins ofthe present invention, as taught herein.

Framework, CDR and hinge regions are labeled in boxes. Sequence residuesare numbered for each amino acid postion. A list of amino acidsubstitutions or gaps (denoted by a “-”) observed at each position inthe aligned sequences are shown below each sequence residue.

FIG. 1 depicts Vh1 heavy chain variable region sequences, frameworks andsubstitutions (SEQ ID NO:31).

FIG. 2 depicts Vh2 heavy chain variable region sequences, frameworks andsubstitutions (SEQ ID NO:32).

FIG. 3 depicts Vh3a heavy chain variable region sequences, frameworksand substitutions (SEQ ID NO:33).

FIG. 4 depicts Vh3b heavy chain variable region sequences, frameworksand substitutions (SEQ ID NO:34).

FIG. 5 depicts Vh3c heavy chain variable region sequences, frameworksand substitutions (SEQ ID NO:35).

FIG. 6 depicts Vh4 heavy chain variable region sequences, frameworks andsubstitutions (SEQ ID NO:36).

FIG. 7 depicts Vh5 heavy chain variable region sequences, frameworks andsubstitutions (SEQ ID NO:37).

FIG. 8 depicts Vh6 heavy chain variable region sequences, frameworks andsubstitutions (SEQ ID NO:38).

FIG. 9 depicts Vh7 heavy chain variable region sequences, frameworks andsubstitutions (SEQ ID NO:39).

FIG. 10 depicts κ1_(—)4 light chain variable region sequences,frameworks and substitutions (SEQ ID NO:40).

FIG. 11 depicts κ2 light chain variable region sequences, frameworks andsubstitutions (SEQ ID NO:41).

FIG. 12 depicts κ3 light chain variable region sequences, frameworks andsubstitutions (SEQ ID NO:42).

FIG. 13 depicts κ5 light chain variable region sequences, frameworks andsubstitutions (SEQ ID NO:43).

FIG. 14 depicts κNew1 light chain variable region sequences, frameworksand substitutions (SEQ ID NO:44).

FIG. 15 depicts κNew2 light chain variable region sequences, frameworksand substitutions (SEQ ID NO:45).

FIG. 16 depicts κNew3 light chain variable region sequences, frameworksand substitutions (SEQ ID NO:46).

FIG. 17 depicts λ1a light chain variable region sequences, frameworksand substitutions (SEQ ID NO:47).

FIG. 18 depicts λ1b light chain variable region sequences, frameworksand substitutions (SEQ ID NO:48).

FIG. 19 depicts λ2 light chain variable region sequences, frameworks andsubstitutions (SEQ ID NO:49).

FIG. 20 depicts λ3a light chain variable region sequences, frameworksand substitutions (SEQ ID NO:50).

FIG. 21 depicts λ3b light chain variable region sequences, frameworksand substitutions (SEQ ID NO:51).

FIG. 22 depicts λ3c light chain variable region sequences, frameworksand substitutions (SEQ ID NO:52).

FIG. 23 depicts λ3e light chain variable region sequences, frameworksand substitutions (SEQ ID NO:53).

FIG. 24 depicts λ4a light chain variable region sequences, frameworksand substitutions (SEQ ID NO:54).

FIG. 25 depicts λ4b light chain variable region sequences, frameworksand substitutions (SEQ ID NO:55).

FIG. 26 depicts λ5 light chain variable region sequences, frameworks andsubstitutions (SEQ ID NO:56).

FIG. 27 depicts λ6 light chain variable region sequences, frameworks andsubstitutions (SEQ ID NO:57).

FIG. 28 depicts λ7 light chain variable region sequences, frameworks andsubstitutions (SEQ ID NO:58).

FIG. 29 depicts λ8 light chain variable region sequences, frameworks andsubstitutions (SEQ ID NO:59).

FIG. 30 depicts λ9 light chain variable region sequences, frameworks andsubstitutions (SEQ ID NO:60).

FIG. 31 depicts λ10 light chain variable region sequences, frameworksand substitutions (SEQ ID NO:61).

FIG. 32 depicts IgA1 heavy chain constant region sequences, subdomainsand substitutions (SEQ ID NO:62).

FIG. 33 depicts IgA2 heavy chain constant region sequences, subdomainsand substitutions (SEQ ID NO:63).

FIG. 34 depicts IgD heavy chain constant region sequences, subdomainsand substitutions (SEQ ID NO:64).

FIG. 35 depicts IgE heavy chain constant region sequences, subdomainsand substitutions (SEQ ID NO:65).

FIG. 36 depicts IgG1 heavy chain constant region sequences, subdomainsand substitutions (SEQ ID NO:66).

FIG. 37 depicts IgG2 heavy chain constant region sequences, subdomainsand substitutions (SEQ ID NO:67).

FIG. 38 depicts IgG3 heavy chain constant region sequences, subdomainsand substitutions (SEQ ID NO:68).

FIG. 39 depicts IgG4 heavy chain constant region sequences, subdomainsand substitutions (SEQ ID NO:69).

FIG. 40 depicts IgM heavy chain constant region sequences, subdomainsand substitutions (SEQ ID NO:70).

FIG. 41 depicts Igκc light chain constant region sequences andsubstitutions (SEQ ID NO:71).

FIG. 42 depicts Igλc light chain constant region sequences andsubstitutions (SEQ ID NO:72).

DESCRIPTION OF THE INVENTION

The present invention provides isolated, recombinant and/or syntheticmimetibodies or specified portions or variants, as well as compositionsand encoding nucleic acid molecules comprising at least onepolynucleotide encoding at least one EPO mimetic hinge core mimetibody.Such mimetibodies or specified portions or variants of the presentinvention comprise specific EPO mimetic hinge core mimetibody sequences,domains, fragments and specified variants thereof, and methods of makingand using said nucleic acids and mimetibodies or specified portions orvariants, including therapeutic compositions, methods and devices.

The present invention also provides at least one isolated EPO mimetichinge core mimetibody or specified portion or variant as describedherein and/or as known in the art. The EPO mimetic hinge core mimetibodycan optionally comprise at least one CH3 region directly linked with atleast one CH2 region directly linked with at least one hinge region orfragment thereof (H), directly linked with an optional linker sequence(L), directly linked to at least one therapeutic peptide (P), optionallyfurther directly linked with at least a portion of at least one variable(V) antibody sequence.

In a preferred embodiment an EPO mimetic hinge core mimetibody comprisesformula (I):((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s),where V is at least one portion of an N-terminus of an immunoglobulinvariable region, P is at least one bioactive peptide, L is polypeptidethat provides structural flexablity by allowing the mimietibody to havealternative orientations and binding properties, H is at least a portionof an immunoglobulin variable hinge region, CH2 is at least a portion ofan immunoglobulin CH2 constant region, CH3 is at least a portion of animmunoglobulin CH3 constant region, m, n, o, p, q, r, and s can beindependently an integer between 0, 1 or 2 and 10, mimicing differenttypes of immunoglobulin molecules, e.g., but not limited to IgG1, IgG2,IgG3, IgG4, IgA, IgM, IgD, IgE, and the like, or combination thereof.The monomer where m=1 can be linked to other monomers by association orcovalent linkage, such as, but not limited to, a Cys-Cys disulfide bondor other immnuoglobulin sequence. EPO mimetic hinge core mimetibody ofthe present invention mimics an antibody structure with its inherentproperties and functions, while providing a therapeutic peptide and itsinherent or acquired in vitro, in vivo or in situ properties oractivities. The various portions of the antibody and therapeutic peptideportions of at least one EPO mimetic hinge core mimetibody of thepresent invention can vary as described herein in combinatoin with whatis known in the art.

As used herein, a “EPO mimetic hinge core mimetibody,” “EPO mimetichinge core mimetibody portion,” or “EPO mimetic hinge core mimetibodyfragment” and/or “EPO mimetic hinge core mimetibody variant” and thelike mimics, has or simulates at least one ligand binding or at leastone biological activity of at least one protein, such as ligand bindingor activity in vitro, in situ and/or preferably in vivo, such as but notlimited to at least one of SEQ ID NOS:1-30. For example, a suitable EPOmimetic hinge core mimetibody, specified portion or variant of thepresent invention can bind at least one protein ligand and includes atleast one protein ligand, receptor, soluble receptor, and the like. Asuitable EPO mimetic hinge core mimetibody, specified portion, orvariant can also modulate, increase, modify, activate, at least oneprotein receptor signaling or other measurable or detectable activity.

Mimetibodies useful in the methods and compositions of the presentinvention are characterized by suitable affinity binding to proteinligands or receptors and optionally and preferably having low toxicity.In particular, an EPO mimetic hinge core mimetibody, where theindividual components, such as the portion of variable region, constantregion (without a CH1 portion) and framework, or any portion thereof(e.g., a portion of the J, D or V rgions of the variable heavy or lightchain; at least a portion of at least one hinge region, the constantheavy chain or light chain, and the like) individually and/orcollectively optionally and preferably possess low immunogenicity, isuseful in the present invention. The mimetibodies that can be used inthe invention are optionally characterized by their ability to treatpatients for extended periods with good to excellent alleviation ofsymptoms and low toxicity. Low immunogenicity and/or high affinity, aswell as other undefined properties, may contribute to the therapeuticresults achieved. “Low immunogenicity” is defined herein as raisingsignificant HAMA, HACA or HAHA responses in less than about 75%, orpreferably less than about 50, 45, 40, 35, 30, 35, 20, 15, 10, 9, 8, 7,6, 5, 4, 3, 2, and/or 1% of the patients treated and/or raising lowtitres in the patient treated (less than about 300, preferably less thanabout 100 measured with a double antigen enzyme immunoassay) (see, e.g.,Elliott et al., Lancet 344:1125-1127 (1994)).

Utility

The isolated nucleic acids of the present invention can be used forproduction of at least one EPO mimetic hinge core mimetibody, fragmentor specified variant thereof, which can be used to effect in an cell,tissue, organ or animal (including mammals and humans), to modulate,treat, alleviate, help prevent the incidence of, or reduce the symptomsof, at least one protein related condition, selected from, but notlimited to, at least one of an immune disorder or disease, acardiovascular disorder or disease, an infectious, malignant, and/orneurologic disorder or disease, a(n) anemia; a(n) immune/autoimmune;and/or a(n) cancer/infecteous, as well as other known or specifiedprotein related conditions.

Such a method can comprise administering an effective amount of acomposition or a pharmaceutical composition comprising at least one EPOmimetic hinge core mimetibody or specified portion or variant to a cell,tissue, organ, animal or patient in need of such modulation, treatment,alleviation, prevention, or reduction in symptoms, effects ormechanisms. The effective amount can comprise an amount of about 0.0001to 500 mg/kg per single or multiple administration, or to achieve aserum concentration of 0.0001-5000 μg/ml serum concentration per singleor multiple adminstration, or any effective range or value therein, asdone and determined using known methods, as described herein or known inthe relevant arts.

Citations

All publications or patents cited herein are entirely incorporatedherein by reference as they show the state of the art at the time of thepresent invention and/or to provide description and enablement of thepresent invention. Publications refer to any scientific or patentpublications, or any other information available in any media format,including all recorded, electronic or printed formats. The followingreferences are entirely incorporated herein by reference: Ausubel, etal., ed., Current Protocols in Molecular Biology, John Wiley & Sons,Inc., NY, N.Y. (1987-2003); Sambrook, et al., Molecular Cloning: ALaboratory Manual, 2^(nd) Edition, Cold Spring Harbor, N.Y. (1989);Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor,N.Y. (1989); Colligan, et al., eds., Current Protocols in Immunology,John Wiley & Sons, Inc., N.Y. (1994-2003); Colligan et al., CurrentProtocols in Protein Science, John Wiley & Sons, NY, N.Y., (1997-2003).

Mimetibodies of the Present Invention

The EPO mimetic hinge core mimetibody can optionally comprise at leastone CH3 region directly linked with at least one CH2 region directlylinked with at least one portion of at lesat one hinge region fragment(H) such as comprising at least one core hinge region, directly linkedwith an optional linker sequence (L), directly linked to at least onetherapeutic peptide (P), optionally further directly linked with atleast a portion of at least one variable antibody sequence (V). In apreferred embodiment a pair of a CH3-CH2-H-L-V, the pair linked byassociation or covalent linkage Thus, an EPO mimetic hinge coremimetibody of the present invention mimics an antibody structure withits inherent properties and functions, while providing a therapeuticpeptide and its inherent or acquired in vitro, in vivo or in situproperties or activities. The various portions of the antibody andtherapeutic peptide portions of at least one EPO mimetic hinge coremimetibody of the present invention can vary as described herein incombinatoin with what is known in the art.

Mimetibodies of the present invention thus provide at least one suitableproperty as compared to known proteins, such as, but not limited to, atleast one of increased half-life, increased activity, more specificactivity, increased avidity, increased or descrease off rate, a selectedor more suitable subset of activities, less immungenicity, increasedquality or duration of at least one desired therapeutic effect, lessside effects, and the like.

Fragments of mimetibodies according to Formula (I) can be produced byenzymatic cleavage, synthetic or recombinant techniques, as known in theart and/or as described herein. Mimetibodies can also be produced in avariety of truncated forms using antibody genes in which one or morestop codons have been introduced upstream of the natural stop site. Thevarious portions of mimetibodies can be joined together chemically byconventional techniques, or can be prepared as a contiguous proteinusing genetic engineering techniques. For example, a nucleic acidencoding at least one of the constant regions of a human antibody chaincan be expressed to produce a contiguous protein for use in mimetibodiesof the present invention. See, e.g., Ladner et al., U.S. Pat. No.4,946,778 and Bird, R. E. et al., Science, 242: 423-426 (1988),regarding single chain antibodies.

As used herein, the term “human mimetibody” refers to an antibody inwhich substantially every part of the protein (e.g., EPO mimeticpeptide, framework, C_(L), C_(H) domains (e.g., C_(H)2, C_(H)3), hinge,(V_(L), V_(H))) is expected to be substantially non-immunogenic inhumans with only minor sequence changes or variations. Such changes orvariations optionally and preferably retain or reduce the immunogenicityin humans relative to non-modified human antibodies, or mimetibodies ofthe prsent invention. Thus, a human antibody and corresponding EPOmimetic hinge core mimetibody of the present invention is distinct froma chimeric or humanized antibody. It is pointed out that a humanantibody and EPO mimetic hinge core mimetibody can be produced by anon-human animal or cell that is capable of expressing humanimmunoglobulins (e.g., heavy chain and/or light chain) genes.

Human mimetibodies that are specific for at least one protein ligand orreceptor thereof can be designed against an appropriate ligand, such asisolated and/or EPO protein receptor or ligand, or a portion thereof(including synthetic molecules, such as synthetic peptides). Preparationof such mimetibodies are performed using known techniques to identifyand characterize ligand binding regions or sequences of at least oneprotein or portion thereof.

In a preferred embodiment, at least one EPO mimetic hinge coremimetibody or specified portion or variant of the present invention isproduced by at least one cell line, mixed cell line, immortalized cellor clonal population of immortalized and/or cultured cells. Immortalizedprotein producing cells can be produced using suitable methods.Preferably, the at least one EPO mimetic hinge core mimetibody orspecified portion or variant is generated by providing nucleic acid orvectors comprising DNA derived or having a substantially similarsequence to, at least one human immunoglobulin locus that isfunctionally rearranged, or which can undergo functional rearrangement,and which further comprises a mimetibody structure as described herein,e.g., but not limited to Formula (I), wherein portions of C- andN-terminal variable regions can be used for V, hinge regions for H, CH2for CH2 and CH3 for CH3, as known in the art.

The term “functionally rearranged,” as used herein refers to a segmentof nucleic acid from an immunoglobulin locus that has undergone V(D)Jrecombination, thereby producing an immunoglobulin gene that encodes animmunoglobulin chain (e.g., heavy chain), or any portion thereof. Afunctionally rearranged immunoglobulin gene can be directly orindirectly identified using suitable methods, such as, for example,nucleotide sequencing, hybridization (e.g., Southern blotting, Northernblotting) using probes that can anneal to coding joints between genesegments or enzymatic amplification of immunoglobulin genes (e.g.,polymerase chain reaction) with primers that can anneal to coding jointsbetween gene segments. Whether a cell produces an EPO mimetic hinge coremimetibody or portion or variant comprising a particular variable regionor a variable region comprising a particular sequence (e.g., at leastone P sequence) can also be determined using suitable methods.

Mimetibodies, specified portions and variants of the present inventioncan also be prepared using at least one EPO mimetic hinge coremimetibody or specified portion or variant encoding nucleic acid toprovide transgenic animals or mammals, such as goats, cows, horses,sheep, and the like, that produce such mimetibodies or specifiedportions or variants in their milk. Such animals can be provided usingknown methods as applied for antibody encoding sequences. See, e.g., butnot limited to, U.S. Pat. Nos. 5,827,690; 5,849,992; 4,873,316;5,849,992; 5,994,616; 5,565,362; 5,304,489, and the like, each of whichis entirely incorporated herein by reference.

Mimetibodies, specified portions and variants of the present inventioncan additionally be prepared using at least one EPO mimetic hinge coremimetibody or specified portion or variant encoding nucleic acid toprovide transgenic plants and cultured plant cells (e.g., but notlimited to tobacco and maize) that produce such mimetibodies, specifiedportions or variants in the plant parts or in cells cultured therefrom.As a non-limiting example, transgenic tobacco leaves expressingrecombinant proteins have been successfully used to provide largeamounts of recombinant proteins, e.g., using an inducible promoter. See,e.g., Cramer et al., Curr. Top. Microbol. Immunol. 240:95-118 (1999) andreferences cited therein. Also, transgenic maize or corn have been usedto express mammalian proteins at commercial production levels, withbiological activities equivalent to those produced in other recombinantsystems or purified from natural sources. See, e.g., Hood et al., Adv.Exp. Med. Biol. 464:127-147 (1999) and references cited therein.Antibodies have also been produced in large amounts from transgenicplant seeds including antibody fragments, such as single chainmimetibodies (scFv's), including tobacco seeds and potato tubers. See,e.g., Conrad et al., Plant Mol. Biol. 38:101-109 (1998) and referencescited therein. Thus, mimetibodies, specified portions and variants ofthe present invention can also be produced using transgenic plants,according to know methods. See also, e.g., Fischer et al., Biotechnol.Appl. Biochem. 30:99-108 (October, 1999), Ma et al., Trends Biotechnol.13:522-7 (1995); Ma et al., Plant Physiol. 109:341-6 (1995); Whitelam etal., Biochem. Soc. Trans. 22:940-944 (1994); and references citedtherein. The above references are entirely incorporated herein byreference.

The mimetibodies of the invention can bind human protein ligands with awide range of affinities (K_(D)). In a preferred embodiment, at leastone human EPO mimetic hinge core mimetibody of the present invention canoptionally bind at least one protein ligand with high affinity. Forexample, at least one EPO mimetic hinge core mimetibody of the presentinvention can bind at least one protein ligand with a K_(D) equal to orless than about 10⁻⁷ M or, more preferably, with a K_(D) equal to orless than about 0.1-9.9 (or any range or value therein) X 10⁻⁷, 10⁻⁸,10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², or 10⁻¹³ M, or any range or value therein.

The affinity or avidity of an EPO mimetic hinge core mimetibody for atleast one protein ligand can be determined experimentally using anysuitable method, e.g., as used for determing antibody-antigen bindingaffinity or avidity. (See, for example, Berzofsky, et al.,“Antibody-Antigen Interactions,” In Fundamental Immunology, Paul, W. E.,Ed., Raven Press: New York, N.Y. (1984); Kuby, Janis Immunology, W. H.Freeman and Company: New York, N.Y. (1992); and methods describedherein). The measured affinity of a particular EPO mimetic hinge coremimetibody-ligand interaction can vary if measured under differentconditions (e.g., salt concentration, pH). Thus, measurements ofaffinity and other ligand-binding parameters (e.g., K_(D), K_(a), K_(d))are preferably made with standardized solutions of EPO mimetic hingecore mimetibody and ligand, and a standardized buffer, such as thebuffer described herein.

Nucleic Acid Molecules

Using the information provided herein, such as the nucleotide sequencesencoding at least 90-100% of the contiguous amino acids of at least oneof SEQ ID NOS:1-30 as well as at least one portion of an antibody,wherein the above sequences are inserted as the P sequence of Formula(I) to provide an EPO mimetic hinge core mimetibody of the presentinvention, further comprising specified fragments, variants or consensussequences thereof, or a deposited vector comprising at least one ofthese sequences, a nucleic acid molecule of the present inventionencoding at least one EPO mimetic hinge core mimetibody or specifiedportion or variant can be obtained using methods described herein or asknown in the art.

Nucleic acid molecules of the present invention can be in the form ofRNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA,including, but not limited to, cDNA and genomic DNA obtained by cloningor produced synthetically, or any combination thereof. The DNA can betriple-stranded, double-stranded or single-stranded, or any combinationthereof. Any portion of at least one strand of the DNA or RNA can be thecoding strand, also known as the sense strand, or it can be thenon-coding strand, also referred to as the anti-sense strand.

Isolated nucleic acid molecules of the present invention can includenucleic acid molecules comprising an open reading frame (ORF),optionally with one or more introns, nucleic acid molecules comprisingthe coding sequence for an EPO mimetic hinge core mimetibody orspecified portion or variant; and nucleic acid molecules which comprisea nucleotide sequence substantially different from those described abovebut which, due to the degeneracy of the genetic code, still encode atleast one EPO mimetic hinge core mimetibody as described herein and/oras known in the art. Of course, the genetic code is well known in theart. Thus, it would be routine for one skilled in the art to generatesuch degenerate nucleic acid variants that code for specific EPO mimetichinge core mimetibody or specified portion or variants of the presentinvention. See, e.g., Ausubel, et al., supra, and such nucleic acidvariants are included in the present invention.

As indicated herein, nucleic acid molecules of the present inventionwhich comprise a nucleic acid encoding an EPO mimetic hinge coremimetibody or specified portion or variant can include, but are notlimited to, those encoding the amino acid sequence of an EPO mimetichinge core mimetibody fragment, by itself; the coding sequence for theentire EPO mimetic hinge core mimetibody or a portion thereof; thecoding sequence for an EPO mimetic hinge core mimetibody, fragment orportion, as well as additional sequences, such as the coding sequence ofat least one signal leader or fusion peptide, with or without theaforementioned additional coding sequences, such as at least one intron,together with additional, non-coding sequences, including but notlimited to, non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences that play a role in transcription, mRNAprocessing, including splicing and polyadenylation signals (forexample—ribosome binding and stability of mRNA); an additional codingsequence that codes for additional amino acids, such as those thatprovide additional functionalities. Thus, the sequence encoding an EPOmimetic hinge core mimetibody or specified portion or variant can befused to a marker sequence, such as a sequence encoding a peptide thatfacilitates purification of the fused EPO mimetic hinge core mimetibodyor specified portion or variant comprising an EPO mimetic hinge coremimetibody fragment or portion.

Polynucleotides Which Selectively Hybridize to a Polynucleotide asDescribed Herein

The present invention provides isolated nucleic acids that hybridizeunder selective hybridization conditions to a polynucleotide disclosedherein, or others disclosed herein, including specified variants orportions thereof. Thus, the polynucleotides of this embodiment can beused for isolating, detecting, and/or quantifying nucleic acidscomprising such polynucleotides.

Low or moderate stringency hybridization conditions are typically, butnot exclusively, employed with sequences having a reduced sequenceidentity relative to complementary sequences. Moderate and highstringency conditions can optionally be employed for sequences ofgreater identity. Low stringency conditions allow selectivehybridization of sequences having about 40-99% sequence identity and canbe employed to identify orthologous or paralogous sequences.

Optionally, polynucleotides of this invention will encode at least aportion of an EPO mimetic hinge core mimetibody or specified portion orvariant encoded by the polynucleotides described herein. Thepolynucleotides of this invention embrace nucleic acid sequences thatcan be employed for selective hybridization to a polynucleotide encodingan EPO mimetic hinge core mimetibody or specified portion or variant ofthe present invention. See, e.g., Ausubel, supra; Colligan, supra, eachentirely incorporated herein by reference.

Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made using(a) recombinant methods, (b) synthetic techniques, (c) purificationtechniques, or combinations thereof, as well-known in the art.

The nucleic acids can conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites can beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences can be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. The nucleic acidof the present invention—excluding the coding sequence—is optionally avector, adapter, or linker for cloning and/or expression of apolynucleotide of the present invention.

Additional sequences can be added to such cloning and/or expressionsequences to optimize their function in cloning and/or expression, toaid in isolation of the polynucleotide, or to improve the introductionof the polynucleotide into a cell. Use of cloning vectors, expressionvectors, adapters, and linkers is well known in the art. See, e.g.,Ausubel, supra; or Sambrook, supra.

Recombinant Methods for Constructing Nucleic Acids

The isolated nucleic acid compositions of this invention, such as RNA,cDNA, genomic DNA, or any combination thereof, can be obtained frombiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probesthat selectively hybridize, under suitable stringency conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library. The isolation of RNA,and construction of cDNA and genomic libraries, is well known to thoseof ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook,supra).

Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the present invention can also be preparedby direct chemical synthesis by known methods (see, e.g., Ausubel, etal., supra). Chemical synthesis generally produces a single-strandedoligonucleotide, which can be converted into double-stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill inthe art will recognize that while chemical synthesis of DNA can belimited to sequences of about 100 or more bases, longer sequences can beobtained by the ligation of shorter sequences.

Recombinant Expression Cassettes

The present invention further provides recombinant expression cassettescomprising a nucleic acid of the present invention. A nucleic acidsequence of the present invention, for example a cDNA or a genomicsequence encoding an EPO mimetic hinge core mimetibody or specifiedportion or variant of the present invention, can be used to construct arecombinant expression cassette that can be introduced into at least onedesired host cell. A recombinant expression cassette will typicallycomprise a polynucleotide of the present invention operably linked totranscriptional initiation regulatory sequences that will direct thetranscription of the polynucleotide in the intended host cell. Bothheterologous and non-heterologous (i.e., endogenous) promoters can beemployed to direct expression of the nucleic acids of the presentinvention.

In some embodiments, isolated nucleic acids that serve as promoter,enhancer, or other elements can be introduced in the appropriateposition (upstream, downstream or in intron) of a non-heterologous formof a polynucleotide of the present invention so as to up or downregulate expression of a polynucleotide of the present invention. Forexample, endogenous promoters can be altered in vivo or in vitro bymutation, deletion and/or substitution, as known in the art. Apolynucleotide of the present invention can be expressed in either senseor anti-sense orientation as desired. It will be appreciated thatcontrol of gene expression in either sense or anti-sense orientation canhave a direct impact on the observable characteristics. Another methodof suppression is sense suppression. Introduction of nucleic acidconfigured in the sense orientation has been shown to be an effectivemeans by which to block the transcription of target genes.

Vectors And Host Cells

The present invention also relates to vectors that include isolatednucleic acid molecules of the present invention, host cells that aregenetically engineered with the recombinant vectors, and the productionof at least one EPO mimetic hinge core mimetibody or specified portionor variant by recombinant techniques, as is well known in the art. See,e.g., Sambrook, et al., supra; Ausubel, et al., supra, each entirelyincorporated herein by reference.

The polynucleotides can optionally be joined to a vector containing aselectable marker for propagation in a host. Generally, a plasmid vectoris introduced into a cell using suitable known methods, such aselectroporation and the like, other known methods include the use of thevector as a precipitate, such as a calcium phosphate precipitate, or ina complex with a charged lipid. If the vector is a virus, it can bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter.The expression constructs will further contain sites optionally for atleast one of transcription initiation, termination and, in thetranscribed region, a ribosome binding site for translation. The codingportion of the mature transcripts expressed by the constructs willpreferably include a translation initiating at the beginning and atermination codon (e.g., UAA, UGA or UAG) appropriately positioned atthe end of the mRNA to be translated, with UAA and UAG preferred formammalian or eukaryotic cell expression.

Expression vectors will preferably but optionally include at least oneselectable marker. Such markers include, e.g., but not limited to,methotrexate (MTX), dihydrofolate reductase (DHFR, U.S. Pat. Nos.4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017,ampicillin, neomycin (G418), mycophenolic acid, or glutamine synthetase(GS, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739) resistance foreukaryotic cell culture, and tetracycline or ampicillin resistance genesfor culturing in E. coli and other bacteria or prokaryotics (the abovepatents are entirely incorporated hereby by reference). Appropriateculture mediums and conditions for the above-described host cells areknown in the art. Suitable vectors will be readily apparent to theskilled artisan. Introduction of a vector construct into a host cell canbe effected by calcium phosphate transfection, DEAE-dextran mediatedtransfection, cationic lipid-mediated transfection, electroporation,transduction, infection or other known methods. Such methods aredescribed in the art, such as Sambrook, supra, Chapters 1-4 and 16-18;Ausubel, supra, Chapters 1, 9, 13, 15, 16.

At least one EPO mimetic hinge core mimetibody or specified portion orvariant of the present invention can be expressed in a modified form,such as a fusion protein, and can include not only secretion signals,but also additional heterologous functional regions. For instance, aregion of additional amino acids, particularly charged amino acids, canbe added to the N-terminus of an EPO mimetic hinge core mimetibody orspecified portion or variant to improve stability and persistence in thehost cell, during purification, or during subsequent handling andstorage. Also, peptide moieties can be added to an EPO mimetic hingecore mimetibody or specified portion or variant of the present inventionto facilitate purification.

Such regions can be removed prior to final preparation of an EPO mimetichinge core mimetibody or at least one fragment thereof. Such methods aredescribed in many standard laboratory manuals, such as Sambrook, supra,Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and18.

Those of ordinary skill in the art are knowledgeable in the numerousexpression systems available for expression of a nucleic acid encoding aprotein of the present invention.

Illustrative of cell cultures useful for the production of themimetibodies, specified portions or variants thereof, are mammaliancells. Mammalian cell systems often will be in the form of monolayers ofcells although mammalian cell suspensions or bioreactors can also beused. A number of suitable host cell lines capable of expressing intactglycosylated proteins have been developed in the art, and include theCOS-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21(e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610, DG44) and BSC-1 (e.g.,ATCC CRL-26) cell lines, hepG2 cells, P3X63Ag8.653, SP2/0-Ag14, 293cells, HeLa cells and the like, which are readily available from, forexample, American Type Culture Collection, Manassas, Va. Preferred hostcells include cells of lymphoid origin such as myeloma and lymphomacells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCCAccession Number CRL-1580) and SP2/0-Ag14 cells (ATCC Accession NumberCRL-1851).

Expression vectors for these cells can include one or more of thefollowing expression control sequences, such as, but not limited to anorigin of replication; a promoter (e.g., late or early SV40 promoters,the CMV promoter (e.g., U.S. Pat. Nos. 5,168,062; 5,385,839), an HSV tkpromoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 alphapromoter (e.g, U.S. Pat. No. 5,266,491), at least one humanimmunoglobulin promoter; an enhancer, and/or processing informationsites, such as ribosome binding sites, RNA splice sites, polyadenylationsites (e.g., an SV40 large T Ag poly A addition site), andtranscriptional terminator sequences. See, e.g., Ausubel et al., supra;Sambrook, et al., supra. Other cells useful for production of nucleicacids or proteins of the present invention are known and/or available,for instance, from the American Type Culture Collection Catalogue ofCell Lines and Hybridomas (www.atcc.org) or other known or commercialsources.

When eukaryotic host cells are employed, polyadenlyation ortranscription terminator sequences are typically incorporated into thevector. An example of a terminator sequence is the polyadenlyationsequence from the bovine growth hormone gene. Sequences for accuratesplicing of the transcript can also be included. An example of asplicing sequence is the VP1 intron from SV40 (Sprague, et al., J.Virol. 45:773-781 (1983)). Additionally, gene sequences to controlreplication in the host cell can be incorporated into the vector, asknown in the art.

Purification of an EPO Mimetic Hinge Core Mimetibody or SpecifiedPortion or Variant Thereof

An EPO mimetic hinge core mimetibody or specified portion or variant canbe recovered and purified from recombinant cell cultures by well-knownmethods including, but not limited to, protein A purification, ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. High performance liquidchromatography (“HPLC”) can also be employed for purification. See,e.g., Colligan, Current Protocols in Immunology, or Current Protocols inProtein Science, John Wiley & Sons, NY, N.Y., (1997-2003), e.g.,Chapters 1, 4, 6, 8, 9, 10, each entirely incorporated herein byreference.

Mimetibodies or specified portions or variants of the present inventioninclude naturally purified products, products of chemical syntheticprocedures, and products produced by recombinant techniques from aeukaryotic host, including, for example, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the EPO mimetic hinge core mimetibody or specifiedportion or variant of the present invention can be glycosylated or canbe non-glycosylated, with glycosylated preferred. Such methods aredescribed in many standard laboratory manuals, such as Sambrook, supra,Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and20, Colligan, Protein Science, supra, Chapters 12-14, all entirelyincorporated herein by reference.

Mimetibodies, Specified Fragments and/or Variants

The isolated mimetibodies of the present invention comprise an EPOmimetic hinge core mimetibody or specified portion or variant encoded byany one of the polynucleotides of the present invention as discussedmore fully herein, or any isolated or prepared EPO mimetic hinge coremimetibody or specified portion or variant thereof.

Preferably, the EPO mimetic hinge core mimetibody or ligand-bindingportion or variant binds at least one EPO protein ligand or receptor,and, thereby provides at least one EPO biological activity of thecorresponding protein or a fragment thereof. Different therapeuticallyor diagnostically significant proteins are well known in the art andsuitable assays or biological activities of such proteins are also wellknown in the art.

Non-limiting examples of suitable EPO mimetic peptides for thisinvention appear in Table 1 below. These peptides can be prepared bymethods disclosed and/or known in the art. Single letter amino acidabbreviations are used in most cases. The X in these sequences (andthroughout this specification, unless specified otherwise in aparticular instance) means that any of the 20 naturally occurring orknown amino acid residues or know derivatives thereof may be present, orany know modified amino acid thereof. Any of these peptides may belinked in tandem (i.e., sequentially), with or without linkers, and afew tandemlinked examples are provided in the table. Linkers are listedas “Δ” and may be any of the linkers described herein. Tandem repeatsand linkers are shown separated by dashes for clarity. Any peptidecontaining a cysteinyl residue may optionally be cross-linked withanother Cys-containing peptide, either or both of which may be linked toa vehicle. A few crosslinked examples are provided in the table. Anypeptide having more than one Cys residue may form an intrapeptidedisulfide bond, as well; see, for example, EPO-mimetic peptides inTable 1. A few examples of intrapeptide disulfide-bonded peptides arespecified in the table. Any of these peptides may be derivatized asdescribed herein, and a few derivatized examples are provided in thetable. For derivatives in which the carboxyl terminus may be capped withan amino group, the capping amino group is shown as —NH₂. Forderivatives in which amino acid residues are substituted by moietiesother than amino acid residues, the substitutions are denoted by a δ,which signifies any of the moieties known in the art, e.g., as describedin Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9 and Cuthbertson etal. (1997), J. Med. Chem. 40:2876-82, which are entirely incorporated byreference. The J substituent and the Z substituents (Z₅, Z₆, . . . Z₄₀)are as defined in U.S. Pat. Nos. 5,608,035, 5,786,331, and 5,880,096,which are entirely incorporated herein by reference. For the EPO-mimeticsequences (Table 1), the substituents X₂ through X₁₁ and the integer “n”are as defined in WO 96/40772, which is entirely incorporated byreference. Residues appearing in boldface are D-amino acids, but can beoptionally L-amino acids. All peptides are linked through peptide bondsunless otherwise noted. Abbreviations are listed at the end of thisspecification. In the “SEQ ID NO.” column, “NR” means that no sequencelisting is required for the given sequence. TABLE 1 EPO-mimetic peptidesequences Sequence/structure SEQ ID NO: YXCXXGPXTWXCXP 1YXCXXGPXTWXCXP-YXCXXGPXTWXCXP 1 YXCXXGPXTWXCXP-Λ-YXCXXGPXTWXCXP 1

1 1 GGTYSCHFGPLTWVCKPQGG 2 GGDYHCRMGPLTWVCKPLGG 3 GGVYACRMGPITWVCSPLGG 4VGNYMCHFGPITWVCRPGGG 5 GGLYLCRFGPVTWDCGYKGG 6 GGTYSCHFGPLTWVCKPQGG 7GGTYSCHFGPLTWVCKPQGG-GGTYSCHFGPLTWVCKPQGG 7GGTYSCHFGPLTWVCKPQGG-Λ-GGTYSCHFGPLTWVCKPQGG 7 GGTYSCHFGPLTWVCKPQGGSSK 8GGTYSCHFGPLTWVCKPQGGSSK-GGTYSCHFGPLTWVCKPQGGSSK 8GGTYSCHFGPLTWVCKPQGGSSK-Λ-GGTYSCHFGPLTWVCKPQGGSSK 8

8 GGTYSCHFGPLTWVCKPQGGSSK(-λ-biotin) 8 CX₄X₅GPX₆TWX₇C 9GGTYSCHGPLTWVCKPQGG 10 VGNYMAHMGPITWVCRPGG 11 GGPHHVYACRMGPLTWIC 12GGTYSCHFGPLTWVCKPQ 13 GGLYACHMGPMTWVCQPLRG 14 TIAQYICYMGPETWECRPSPKA 15YSCHFGPLTWVCK 16 YCHFGPLTWVC 17 X₃X₄X₅GPX₆TWX₇X₈ 18 YX₂X₃X₄X₅GPX₆TWX₇X₈19 X₁YX₂X₃X₄X₅GPX₆X₇X₈X₉X₁₀X₁₁ 20 X₁YX₂CX₄X₅GPX₆TWX₇CX₉X₁₀X₁₁ 21GGLYLCRFGPVTWDCGYKGG 22 GGTYSCHFGPLTWVCKPQGG 23 VGNYMCHFGPITWVCRPGGG 24GGVYACRMGPITWVCSPLGG 25 TIAQYICYMGPETWECRPSPKA 26 YSCHFGPLTWVCK 27YCHFGPLTWVC 28 SCHFGPLTWVCK 29 (AX₂)_(n)X₃X₄X₅GPX₆TWX₇X₈ 30

EPO biological activities are well known in the art. See, e.g.,Anagnostou A et al Erythropoietin has a mitogenic and positivechemotactic effect on endothelial cells. Proceedings of the NationalAcademy of Science (USA) 87: 5978-82 (1990); Fandrey J and Jelkman W EInterleukin 1 and tumor necrosis factor-alpha inhibit erythropoietinproduction in vitro. Annals of the New York Academy of Science 628:250-5 (1991); Geissler K et al Recombinant human erythropoietin: Amultipotential hemopoietic growth factor in vivo and in vitro. Contrib.Nephrol. 87: 1-10 (1990); Gregory C J Erythropoietin sensitivity as adifferentiation marker in the hemopoietic system. Studies of threeerythropoietic colony responses in culture. Journal of CellularPhysiology 89: 289-301 (1976); Jelkman W et al Monokines inhibitingerythropoietin production in human hepatoma cultures and in isolatedperfused rat kidneys. Life Sci. 50: 301-8 (1992); Kimata H et al Humanrecombinant erythropoietin directly stimulates B cell immunoglobulinproduction and proliferation in serum-free medium. Clinical andExperimental Immunology 85: 151-6 (1991); Kimata H et al Erythropoietinenhances immunoglobulin production and proliferation by human plasmacells in a serum-free medium. Clin. Immunology Immunopathol. 59: 495-501(1991); Kimata H et al Effect of recombinant human erythropoietin onhuman IgE production in vitro Clinical and Experimental Immunology 83:483-7 (1991); Koury M J and Bondurant M C Erythropoietin retards DNAbreakdown and prevents programmed cell death in erythroid progenitorcells. Science 248: 378-81 (1990); Lim V S et al Effect of recombinanthuman erythropoietin on renal function in humans. Kidney International37: 131-6 (1990); Mitjavila M T et al Autocrine stimulation byerythropoietin and autonomous growth of human erythroid leukemic cellsin vitro. Journal of Clinical Investigation 88: 789-97 (1991); Andre Met al Performance of an immunoradiometric assay of erythropoietin andresults for specimens from anemic and polycythemic patients. ClinicalChemistry 38: 758-63 (1992); Hankins W D et al Erythropoietin-dependentand erythropoietin-producing cell lines. Implications for research andfor leukemia therapy. Annals of the New York Academy of Science 554:21-8 (1989); Kendall R G T et al Storage and preparation of samples forerythropoietin radioimmunoassay. Clin. Lab. Haematology 13: 189-96(1991); Krumvieh D et al Comparison of relevant biological assays forthe determination of biological active erythropoietin. Dev. Biol. Stand.69: 15-22 (1988); Ma D D et al Assessment of an EIA for measuring humanserum erythropoietin as compared with RIA and an in-vitro bioassay.British Journal of Haematology 80: 431-6 (1992); Noe G et al A sensitivesandwich ELISA for measuring erythropoietin in human serum BritishJournal of Haematology 80: 285-92 (1992); Pauly J U et al Highlyspecific and highly sensitive enzyme immunoassays for antibodies tohuman interleukin 3 (IL3) and human erythropoietin (EPO) in serum.Behring Institut Mitteilungen 90: 112-25 (1991); Sakata S and Enoki YImproved microbioassay for plasma erythropoietin based on CFU-E colonyformation. Ann. Hematology 64: 224-30 (1992); Sanengen T et alImmunoreactive erythropoietin and erythropoiesis stimulating factor(s)in plasma from hypertransfused neonatal and adult mice.

Studies with a radioimmunoassay and a cell culture assay forerythropoietin. Acta Physiol. Scand. 135: 11-6 (1989); Widness J A et alA sensitive and specific erythropoietin immunoprecipitation assay:application to pharmacokinetic studies. Journal of Lab. Clin. Med. 119:285-94 (1992); for further information see also individual cell linesused in individual bioassays. Each of the above references are entirelyincorporated herein by reference. EPO can be assayed by employing celllines such as HCD57, NFS-60, TF-1 and UT-7 , which respond to thefactor. EPO activity can be assessed also in a Colony formation assay bydetermining the number of CFU-E from bone marrow cells. An alternativeand entirely different detection method is RT-PCR quantitation ofcytokines.

An EPO mimetic hinge core mimetibody, or specified portion or variantthereof, that partially or preferably substantially provides at leastone biological activity of at least one protein or fragment, can bindthe protein or fragment ligand and thereby provide at least one activitythat is otherwise mediated through the binding of protein to at leastone protein ligand or receptor or through other protein-dependent ormediated mechanisms. As used herein, the term “EPO mimetic hinge coremimetibody activity” refers to an EPO mimetic hinge core mimetibody thatcan modulate or cause at least one protein-dependent activity by about20-10,000%, preferably by at least about 60, 70, 80, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 2000,3000, 4000, 5000, 6000, 7000, 8000, 9000% or more depending on theassay.

The capacity of an EPO mimetic hinge core mimetibody or specifiedportion or variant to provide at least one protein-dependent activity ispreferably assessed by at least one suitable protein biological assay,as described herein and/or as known in the art. A human EPO mimetichinge core mimetibody or specified portion or variant of the inventioncan be similar to any class (IgG, IgA, IgM, etc.) or isotype and cancomprise at least a portion of a kappa or lambda light chain. In oneembodiment, the human EPO mimetic hinge core mimetibody or specifiedportion or variant comprises an IgG heavy chain variable fragment, hingeregion, CH2 and CH3, for example, at least one of isotypes, IgG1, IgG2,IgG3 or IgG4.

At least one EPO mimetic hinge core mimetibody or specified portion orvariant of the invention binds at least one specified ligand specific toat least one protein, subunit, fragment, portion or any combinationthereof. The at least one EPO mimeiic peptide of at least one EPOmimetic hinge core mimetibody, specified portion or variant of thepresent invention can optionally bind at least one specified ligandepitope of the ligand. The binding epitope can comprise any combinationof at least one amino acid sequence of at least 1-3 amino acids to theentire specified portion of contiguous amino acids of the sequencesselected from the group consisting of a protein ligand, such as an EPOreceptorz or portion thereof.

Such mimetibodies can be prepared by joining together the variousportions of Formula (I) of the EPO mimetic hinge core mimetibody usingknown techniques, by preparing and expressing at least one (i.e., one ormore) nucleic acid molecules that encode the EPO mimetic hinge coremimetibody, using known techniques of recombinant DNA technology or byusing any other suitable method, such as chemical synthesis.

Mimetibodies that bind to human EPO ligands or receptors and thatcomprise at least a one portion defined heavy or light chain variableregion can be prepared using suitable methods, such as phage display(Katsube, Y., et al., Int J Mol. Med, 1(5):863-868 (1998)) or methodsthat employ transgenic animals, as known in the art and/or as describedherein. The EPO mimetic hinge core mimetibody, specified portion orvariant can be expressed using the encoding nucleic acid or portionthereof in a suitable host cell.

Preferably, such mimetibodies or ligand-binding fragments thereof canbind human EPO ligands or receptors with high affinity (e.g., K_(D) lessthan or equal to about 10⁻⁷ M). Amino acid sequences that aresubstantially the same as the sequences described herein includesequences comprising conservative amino acid substitutions, as well asamino acid deletions and/or insertions. A conservative amino acidsubstitution refers to the replacement of a first amino acid by a secondamino acid that has chemical and/or physical properties (e.g, charge,structure, polarity, hydrophobicity/hydrophilicity) that are similar tothose of the first amino acid. Conservative substitutions includereplacement of one amino acid by another within the following groups:lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate(E); asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine(Y), K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine(I), proline (P), phenylalanine (F), tryptophan (W), methionine (M),cysteine (C) and glycine (G); F, W and Y; C, S and T.

Amino Acid Codes

The amino acids that make up mimetibodies or specified portions orvariants of the present invention are often abbreviated. The amino aciddesignations can be indicated by designating the amino acid by itssingle letter code, its three letter code, name, or three nucleotidecodon(s) as is well understood in the art (see Alberts, B., et al.,Molecular Biology of The Cell, Third Ed., Garland Publishing, Inc.,NewYork, 1994), as presented in Table TABLE 2 SINGLE THREE THREE LETTERLETTER NUCLEOTIDE CODE CODE NAME CODON(S) A Ala Alanine GCA, GCC, GCG,GCU C Cys Cysteine UGC, UGU D Asp Aspartic acid GAC, GAU E Glu Glutamicacid GAA, GAG F Phe Phenylanine UUC, UUU G Gly Glycine GGA, GGC, GGG,GGU H His Histidine CAC, CAU I Ile Isoleucine AUA, AUC, AUU K Lys LysineAAA, AAG L Leu Leucine UUA, UUG, CUA, CUC, CUG, CUU M Met Methionine AUGN Asn Asparagine AAC, AAU P Pro Proline CCA, CCC, CCG, CCU Q GlnGlutamine CAA, CAG R Arg Arginine AGA, AGG, CGA, CGC, CGG, CGU S SerSerine AGC, AGU, UCA, UCC, UCG, UCU T Thr Threonine ACA, ACC, ACG, ACU VVal Valine GUA, GUC, GUG, GUU W Trp Tryptophan UGG Y Tyr Tyrosine UAC,UAU

An EPO mimetic hinge core mimetibody or specified portion or variant ofthe present invention can include one or more amino acid substitutions,deletions or additions, either from natural mutations or humanmanipulation, as specified herein. Such or other sequences that can beused in the present invention, include, but are not limited to thefollowing sequences presented in Table 3, as further described in FIGS.1-42, with-corresponding SEQ ID NOS31-72. TABLE 3 SEQ ID AA REGIONS NONO FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 31 Heavy chain Vh1 125 1-31 32 33-4647 48-79 80  81-125 32 variable Vh2 124 1-30 31 32-45 46 47-78 79 80-124 33 region Vh3a 100 1-31 32 33-46 47 48-79 80  81-100 34 Vh3b 1021-30 31 32-45 46 47-78 79  80-102 35 Vh3c 101 1-30 31 32-45 46 47-79 80 81-101 36 Vh4 108 1-33 34 35-48 49 50-81 82  83-108 37 Vh5 132 1-31 3233-46 47 48-79 80  81-132 38 Vh6 125 1-30 31 32-45 46 47-78 79  80-12539 Vh7 91 1-30 31 32-45 46 47-78 79 80-91 40 Light chain κ1-4 93 1-24 2526-40 41 42-73 74 75-93 41 variable κ2 92 1-23 24 25-39 40 41-72 7374-92 42 region κ3 91 1-23 24 25-39 40 41-72 73 74-91 43 κ5 85 1-23 2425-39 40 41-72 73 74-85 44 κ new1 79 1-17 18 19-33 34 35-66 67 68-79 45κ new2 77 1-15 16 17-31 32 33-64 65 66-77 46 κ new3 95 1-24 25 26-40 4142-73 74 75-95 47 λ1a 98 1-22 23 24-38 39 40-71 72 73-98 48 λ1b 99 1-2324 25-39 40 41-72 73 74-99 49 λ2 99 1-22 23 24-38 39 40-71 72 73-99 50λ3a 107 1-22 23 24-38 39 40-71 72  73-107 51 λ3b 93 1-22 23 24-39 4041-72 73 74-93 52 λ3c 98 1-22 23 24-38 39 40-71 72 73-98 53 λ3e 98 1-2223 24-38 39 40-71 72 73-98 54 λ4a 94 1-22 23 24-38 39 40-71 72 73-94 55λ4b 95 1-22 23 24-38 39 40-71 72 73-95 56 λ5 88 1-22 23 24-39 40 41-7475 76-88 57 λ6 101 1-22 23 24-38 39 40-73 74  75-101 58 λ7 89 1-22 2324-38 39 40-71 72 73-89 59 λ8 89 1-22 23 24-38 39 40-71 72 73-89 60 λ991 1-22 23 24-38 39 40-79 80 81-91 61 λ10 87 1-22 23 24-38 39 40-71 7273-87 SEQ ID AA REGIONS NO NO CH1 hinge1 hinge2 hinge3 hinge4 CH2 CH3 62Heavy chain IgA1 354 1-102 103-121  122-222 223-354 63 constant IgA2 3401-102 103-108  109-209 210-340 64 region IgD 384 1-101 102-135  136-159160-267 268-384 65 IgE 497 1-103 104-210 211-318 66 IgG1 339 1-98 99-113 114-223 224-339 67 IgG2 326 1-98  99-110 111-219 220-326 68 IgG3377 1-98  99-115 116-130 131-145 146-160 161-270 271-377 69 IgG4 3271-98  99-110 111-220 221-327 70 IgM 476 1-104 105-217 218-323 71 Lightchain Igκc 107 72 constant Igλc 107 region

Of course, the number of amino acid substitutions a skilled artisanwould make depends on many factors, including those described above.Generally speaking, the number of amino acid substitutions, insertionsor deletions for at least one of an EPO mimetic hinge core mimetibodywill not be more than 40, 30, 20,19, 18, 17, 16, 15, 14, 13, 12, 11, 10,9, 8, 7, 6, 5, 4, 3, 2, 1 amino acids, such as 1-30 or any range orvalue therein, as specified herein.

The following description of the components of an EPO hinge coremimetibody of the present invention is based on the use of the formula Iof the present invention,((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s),

where V is at least one portion of an N-terminus of an immunoglobulinvariable region, P is at least one bioactive peptide, L is at least onelinker polypeptide H is at least one portion of at least oneimmunoglobulin hinge region, CH2 is at least a portion of animmunoglobulin CH2 constant region, CH3 is at least a portion of animmunoglobulin CH3 constant region, m, n, o, p, q, r and s areindependently an integer between 0, 1 or 2 and 10, mimicing differenttypes of immunoglobulin molecules, e.g., but not limited to IgG1, IgG2,IgG3, IgG4, IgA, IgM, IgD, IgE, and the like, or any subclass thereof,or any combination thereof.

In hinge core mimetibodies of the present invention, the optionalN-terminal V portion can comprise 1-20 amino acids of at least one heavychain variable framework 1 (FR1) region, e.g., as presented in FIGS. 1-9(SEQ ID NOS:31-39) or at least one LC variable region, e.g., aspresented in FIGS. 10-31 (SEQ ID NOS:40-61), including substitutions,deletions or insertions as presented in these Figures, with those ofFIGS. 5, 6, and 8 preferred. Also preferred are variable sequences thatcomprise the sequence Q-X-Q.

The P portion can comprise at least one any therapeutic peptide as knownin the art or as described herein, such as, but not limited to thosepresented in Table 1, SEQ ID NOS:1-30, or as known in the art, or anycombination or consensus sequence thereof, or any fusion proteinthereof.

The optional linker sequence can be any suitable peptide linker as knownin the art. Preferred sequence include any combination of G and S, e.g.,X1-X2-X3-X4-Xn, where X can be G or S, and n can be 5-30. Non-limitingexamples include, GS, GGGS, GSGGGS, GSGGGSGG, and the like.

In the present invention, the CH1 portion is not used and a variablenumber of amino acids from the N-terminus of the hinge region aredeleted, e.g., as referenced to FIGS. 1-42 and Table 3. The variablenumber of amino acids used for the hinge core portion of a mimetibody ofthe present invention include, but are not limited to, deletion of anyof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, or 1-3, 2-5, 2-7, 2-8, 3-9, 4-10, 5-9, 5-10, 5-15, 10-20, 2-30,20-40, 10-50, or any range or value therein, of the N-terminal aminoacids of at least one hinge region, e.g., as presented in FIGS. 32-40,or Table 3 above, e.g., but not limited to, deletion of any to all ofthe amino acids 99-101 to 105-157 of amino acids 99-105, 99-108, 99-111,99-112, 99-113, 99-114, 99-115, 99-119, 99-125, 99-128, 99-134, 99-140,99-143, 99-149, 99-155 and 99-158 of FIGS. 32-40, corresponding to SEQID NOS:62-70, including the substitutions, insertions or deletionsdescribed in FIGS. 32-40. In preferred embodiments, a hinge core regionsof the present invention includes a deletion of the N-terminous of thehinge region to provide a hinge core region that includes a deletion upto but not including a Cys residue or up to but not including a sequenceCys-Pro-Xaa-Cys. In further preferred embodiment, such hinge coresequences used in a hinge core mimetibody of the present inventioninclude amino acids 109-113 or 112-113 of FIG. 36 (SEQ ID NO:66) (IgG1);105-110 or 109-110 of FIG. 37 (SEQ ID NO:67) (IgG2); 111-160, 114-160,120-160, 126-160, 129-160, 135-160, 141-160, 144-160, 150-160, 156-160and 159-160 of FIG. 38 (SEQ ID NO:68) (IgG3); or 106-110 or 109-110 ofFIG. 39 (SEQ ID NO:69) (IgG4).

The CH2, CH3 and optional CH4 sequence can be any suitable human orhuman compatable sequence, e.g., as presented in FIGS. 1-42 and Table 3,or as known in the art, or any combination or consensus sequencethereof, or any fusion protein thereof.

Amino acids in an EPO mimetic hinge core mimetibody or specified portionor variant of the present invention that are essential for function canbe identified by methods known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (e.g., Ausubel, supra,Chapters 8, 15; Cunningham and Wells, Science 244:1081-1085 (1989)). Thelatter procedure introduces single alanine mutations at every residue inthe molecule. The resulting mutant molecules are then tested forbiological activity, such as, but not limited to at least one proteinrelated activity, as specified herein or as known in the art. Sites thatare critical for EPO mimetic hinge core mimetibody or specified portionor variant binding can also be identified by structural analysis such ascrystallization, nuclear magnetic resonance or photoaffinity labeling(Smith, et al., J. Mol. Biol. 224:899-904 (1992) and de Vos, et al.,Science 255:306-312 (1992)).

Mimetibodies or specified portions or variants of the present inventioncan comprise as the P portion of Formula (I), but are not limited to, atleast one portion, sequence or combination selected from 3 to all the ofat least one of SEQ ID NOS:1-30. Non-limiting variants that can enhanceor maintain at least one of the listed activities above include, but arenot limited to, any of the above polypeptides, further comprising atleast one mutation corresponding to at least one substitution, insertionor deletion that does not significantly affect the suitable biologicalactivtities or functions of said EPO mimetic hinge core mimetibody.

An EPO mimetic hinge core mimetibody or specified portion or variant canfurther optionally comprise at least one functional portion of at leastone polypeptide as P portion of Formula (I), at least one of 90-100% ofSEQ ID NOS:1-30. An EPO mimetic hinge core mimetibody can furtheroptionally comprise an amino acid sequence for the P portion of Formula(I), selected from one or more of SEQ ID NOS:1-30.

In one embodiment, the P amino acid sequence, or portion thereof hasabout 90-100% identity (i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100 or any range or value therein) to the corresponding amino acidsequence of the corresponding portion of at least one of SEQ IDNOS:1-30. Preferably, 90-100% amino acid identity (i.e., 90, 91, 92, 93,94,95, 96, 97, 98, 99, 100 or any range or value therein) is determinedusing a suitable computer algorithm, as known in the art.

Mimetibodies or specified portions or variants of the present inventioncan comprise any number of contiguous amino acid residues from an EPOmimetic hinge core mimetibody or specified portion or variant of thepresent invention, wherein that number is selected from the group ofintegers consisting of from 10-100% of the number of contiguous residuesin an EPO mimetic hinge core mimetibody. Optionally, this subsequence ofcontiguous amino acids is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250 or more amino acids in length, or anyrange or value therein. Further, the number of such subsequences can beany integer selected from the group consisting of from 1 to 20, such asat least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, or more.

As those of skill will appreciate, the present invention includes atleast one biologically active EPO mimetic hinge core mimetibody orspecified portion or variant of the present invention. Biologicallyactive mimetibodies or specified portions or variants have a specificactivity at least 20%, 30%, or 40%, and preferably at least 50%, 60%, or70%, and most preferably at least 80%, 90%, or 95%-1000% of that of thenative (non-synthetic), endogenous or related and known inserted orfused protein or specified portion or variant. Methods of assaying andquantifying measures of enzymatic activity and substrate specificity,are well known to those of skill in the art.

In another aspect, the invention relates to human mimetibodies andligand-binding fragments, as described herein, which are modified by thecovalent attachment of an organic moiety. Such modification can producean EPO mimetic hinge core mimetibody or ligand-binding fragment withimproved pharmacokinetic properties (e.g., increased in vivo serumhalf-life). The organic moiety can be a linear or branched hydrophilicpolymeric group, fatty acid group, or fatty acid ester group. Inparticular embodiments, the hydrophilic polymeric group can have amolecular weight of about 800 to about 120,000 Daltons and can be apolyalkane glycol (e.g., polyethylene glycol (PEG), polypropylene glycol(PPG)), carbohydrate polymer, amino acid polymer or polyvinylpyrolidone, and the fatty acid or fatty acid ester group can comprisefrom about eight to about forty carbon atoms.

The modified mimetibodies and ligand-binding fragments of the inventioncan comprise one or more organic moieties that are covalently bonded,directly or indirectly, to the EPO mimetic hinge core mimetibody orspecified portion or variant. Each organic moiety that is bonded to anEPO mimetic hinge core mimetibody or ligand-binding fragment of theinvention can independently be a hydrophilic polymeric group, a fattyacid group or a fatty acid ester group. As used herein, the term “fattyacid” encompasses mono-carboxylic acids and di-carboxylic acids. A“hydrophilic polymeric group,” as the term is used herein, refers to anorganic polymer that is more soluble in water than in octane. Forexample, polylysine is more soluble in water than in octane. Thus, anEPO mimetic hinge core mimetibody modified by the covalent attachment ofpolylysine is encompassed by the invention. Hydrophilic polymerssuitable for modifying mimetibodies of the invention can be linear orbranched and include, for example, polyalkane glycols (e.g., PEG,monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates(e.g., dextran, cellulose, oligosaccharides, poiysaccharides and thelike), polymers of hydrophilic amino acids (e.g., polylysine,polyarginine, polyaspartate and the like), polyalkane oxides (e.g.,polyethylene oxide, polypropylene oxide and the like) and polyvinylpyrolidone. Preferably, the hydrophilic polymer that modifies the EPOmimetic hinge core mimetibody of the invention has a molecular weight ofabout 800 to about 150,000 Daltons as a separate molecular entity. Forexample, PEG₂₅₀₀, PEG₅₀₀₀, PEG₇₅₀₀, PEG₉₀₀₀, PEG₁₀₀₀₀ PEG₁₂₅₀₀,PEG₁₅₀₀₀, and PEG_(20,000), wherein the subscript is the averagemolecular weight of the polymer in Daltons, can be used.

The hydrophilic polymeric group can be substituted with one to about sixalkyl, fatty acid or fatty acid ester groups. Hydrophilic polymers thatare substituted with a fatty acid or fatty acid ester group can beprepared by employing suitable methods. For example, a polymercomprising an amine group can be coupled to a carboxylate of the fattyacid or fatty acid ester, and an activated carboxylate (e.g., activatedwith N,N-carbonyl diimidazole) on a fatty acid or fatty acid ester canbe coupled to a hydroxyl group on a polymer.

Fatty acids and fatty acid esters suitable for modifying mimetibodies ofthe invention can be saturated or can contain one or more units ofunsaturation. Fatty acids that are suitable for modifying mimetibodiesof the invention include, for example, n-dodecanoate (C₁₂, laurate),n-tetradecanoate (C₁₄, myristate), n-octadecanoate (C₁₈, stearate),n-eicosanoate (C₂₀, arachidate), n-docosanoate (C₂₂, behenate),n-triacontanoate (C₃₀), n-tetracontanoate (C₄₀), cis-Δ9-octadecanoate(C₁₈, oleate), all cis-Δ5,8,1 1,14-eicosatetraenoate (C₂₀,arachidonate), octanedioic acid, tetradecanedioic acid, octadecanedioicacid, docosanedioic acid, and the like.

Suitable fatty acid esters include mono-esters of dicarboxylic acidsthat comprise a linear or branched lower alkyl group. The lower alkylgroup can comprise from one to about twelve, preferably one to aboutsix, carbon atoms.

The modified human mimetibodies and ligand-binding fragments can beprepared using suitable methods, such as by reaction with one or moremodifying agents. A “modifying agent” as the term is used herein, refersto a suitable organic group (e.g., hydrophilic polymer, a fatty acid, afatty acid ester) that comprises an activating group. An “activatinggroup” is a chemical moiety or functional group that can, underappropriate conditions, react with a second chemical group therebyforming a covalent bond between the modifying agent and the secondchemical group. For example, amine-reactive activating groups includeelectrophilic groups such as tosylate, mesylate, halo (chloro, bromo,fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like.Activating groups that can react with thiols include, for example,maleimide, iodoacetyl, acrylolyl, pyridyl disulfides,5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. An aldehydefunctional group can be coupled to amine- or hydrazide-containingmolecules, and an azide group can react with a trivalent phosphorousgroup to form phosphoramidate or phosphorimide linkages. Suitablemethods to introduce activating groups into molecules are known in theart (see for example, Hermanson, G. T., Bioconjugate Techniques,Academic Press: San Diego, Calif. (1996)). An activating group can bebonded directly to the organic group (e.g., hydrophilic polymer, fattyacid, fatty acid ester), or through a linker moiety, for example adivalent C₁-C₁₂ group wherein one or more carbon atoms can be replacedby a heteroatom such as oxygen, nitrogen or sulfur. Suitable linkermoieties include, for example, tetraethylene glycol, —(CH₂)₃—,—NH—(CH₂)₆—NH—, —(CH₂)₂—NH— and —CH₂—O—CH₂—CH₂—O—CH₂—CH₂—O—CH—NH—.Modifying agents that comprise a linker moiety can be produced, forexample, by reacting a mono-Boc-alkyldiamine (e.g.,mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid inthe presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) toform an amide bond between the free amine and the fatty acidcarboxylate. The Boc protecting group can be removed from the product bytreatment with trifluoroacetic acid (TFA) to expose a primary amine thatcan be coupled to another carboxylate as described, or can be reactedwith maleic anhydride and the resulting product cyclized to produce anactivated maleimido derivative of the fatty acid. (See, for example,Thompson, et al., WO 92/16221 the entire teachings of which areincorporated herein by reference.)

The modified mimetibodies of the invention can be produced by reactingan human EPO mimetic hinge core mimetibody or ligand-binding fragmentwith a modifying agent. For example, the organic moieties can be bondedto the EPO mimetic hinge core mimetibody in a non-site specific mannerby employing an amine-reactive modifying agent, for example, an NHSester of PEG. Modified human mimetibodies or ligand-binding fragmentscan also be prepared by reducing disulfide bonds (e.g., intra-chaindisulfide bonds) of an EPO mimetic hinge core mimetibody orligand-binding fragment. The reduced EPO mimetic hinge core mimetibodyor ligand-binding fragment can then be reacted with a thiol-reactivemodifying agent to produce the modified EPO mimetic hinge coremimetibody of the invention. Modified human mimetibodies andligand-binding fragments comprising an organic moiety that is bonded tospecific sites of an EPO mimetic hinge core mimetibody or specifiedportion or variant of the present invention can be prepared usingsuitable methods, such as reverse proteolysis (Fisch et al.,Bioconjugate Chem., 3:147-153 (1992); Werlen et al., Bioconjugate Chem.,5:411-417 (1994); Kumaran et al., Protein Sci. 6(10):2233-2241 (1997);Itoh et al., Bioorg. Chem., 24(1): 59-68 (1996); Capellas et al.,Biotechnol. Bioeng., 56(4):456-463 (1997)), and the methods described inHermanson, G. T., Bioconjugate Techniques, Academic Press: San Diego,Calif. (1996).

EPO Mimetic Hinge Core Mimetibody Compositions

The present invention also provides at least one EPO mimetic hinge coremimetibody or specified portion or variant composition comprising atleast one, at least two, at least three, at least four, at least five,at least six or more mimetibodies or specified portions or variantsthereof, as described herein and/or as known in the art that areprovided in a non-naturally occurring composition, mixture or form. Suchcomposition percentages are by weight, volume, concentration, molarity,or molality as liquid or dry solutions, mixtures, suspension, emulsionsor colloids, as known in the art or as described herein.

Such compositions can comprise 0.00001-99.9999 percent by weight,volume, concentration, molarity, or molality as liquid, gas, or drysolutions, mixtures, suspension, emulsions or colloids, as known in theart or as described herein, on any range or value therein, such as butnot limited to 0.00001, 0.00003, 0.00005, 0.00009, 0.0001, 0.0003,0.0005, 0.0009, 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05,0.09, 0.1, 0.2, 0.3, 0.4., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7,2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3,4.5, 4.6, 4.7, 4.8, 4.9, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1,99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 35 99.9%. Such compositions ofthe present invention thus include but are not limited to 0.00001-100mg/ml and/or 0.00001-100 mg/g.

The composition can optionally further comprise an effective amount ofat least one compound or protein selected from at least one of ananti-infective drug, a cardiovascular (CV) system drug, a centralnervous system (CNS) drug, an autonomic nervous system (ANS) drug, arespiratory tract drug, a gastrointestinal (GI) tract drug, a hormonaldrug, a drug for fluid or electrolyte balance, a hematologic drug, anantineoplactic, an immunomodulation drug, an ophthalmic, otic or nasaldrug, a topical drug, a nutritional drug or the like. Such drugs arewell known in the art, including formulations, indications, dosing andadministration for each presented herein (see., e.g., Nursing 2001Handbook of Drugs, 21^(st) edition, Springhouse Corp., Springhouse, Pa.,2001; Health Professional's Drug Guide 2001, ed., Shannon, Wilson,Stang, Prentice-Hall, Inc, Upper Saddle River, N.J.; PharmcotherapyHandbook, Wells et al., ed., Appleton & Lange, Stamford, Conn., eachentirely incorporated herein by reference).

The anti-infective drug can be at least one selected from amebicides orat least one antiprotozoals, anthelmintics, antifungal s, antimalarials,antituberculotics or at least one antileprotics, aminoglycosides,penicillins, cephalosporins, tetracyclines, sulfonamides,fluoroquinolones, antivirals, macrolide anti-infectives, miscellaneousanti-infectives. The CV drug can be at least one selected frominotropics, antiarrhythmics, antianginals, antihypertensives,antilipemics, miscellaneous cardiovascular drugs. The CNS drug can be atleast one selected from nonnarcotic analgesics or at least one selectedfrom antipyretics, nonsteroidal anti-inflammatory drugs, narcotic or atleast one opiod analgesics, sedative-hypnotics, anticonvulsants,antidepressants, antianxiety drugs, antipsychotics, central nervoussystem stimulants, antiparkinsonians, miscellaneous central nervoussystem drugs. The ANS drug can be at least one selected fromcholinergics (parasympathomimetics), anticholinergics, adrenergics(sympathomimetics), adrenergic blockers (sympatholytics), skeletalmuscle relaxants, neuromuscular blockers. The respiratory tract drug canbe at least one selected from antihistamines, bronchodilators,expectorants or at least one antitussives, miscellaneous respiratorydrugs. The GI tract drug can be at least one selected from antacids orat least one adsorbents or at least one antiflatulents, digestiveenzymes or at least one gallstone solubilizers, antidiarrheals,laxatives, antiemetics, antiulcer drugs. The hormonal drug can be atleast one selected from corticosteroids, androgens or at least oneanabolic steroids, estrogens or at least one progestins, gonadotropins,antidiabetic drugs or at least one glucagon, thyroid hormones, thyroidhormone antagonists, pituitary hormones, parathyroid-like drugs. Thedrug for fluid and electrolyte balance can be at least one selected fromdiuretics, electrolytes or at least one replacement solutions,acidifiers or at least one alkalinizers. The hematologic drug can be atleast one selected from hematinics, anticoagulants, blood derivatives,thrombolytic enzymes. The antineoplastics can be at least one selectedfrom alkylating drugs, antimetabolites, antibiotic antineoplastics,antineoplastics that alter hormone balance, miscellaneousantineoplastics. The immunomodulation drug can be at least one selectedfrom immunosuppressants, vaccines or at least one toxoids, antitoxins orat least one antivenins, immune serums, biological response modifiers.The ophthalmic, otic, and nasal drugs can be at least one selected fromophthalmic anti-infectives, ophthalmic anti-inflammatories, miotics,mydriatics, ophthalmic vasoconstrictors, miscellaneous ophthalmics,otics, nasal drugs. The topical drug can be at least one selected fromlocal anti-infectives, scabicides or at least one pediculicides, topicalcorticosteroids. The nutritional drug can be at least one selected fromvitamins, minerals, or calorics. See, e.g., contents of Nursing 2001Drug Handbook, supra.

The at least one amebicide or antiprotozoal can be at least one selectedfrom atovaquone, chloroquine hydrochloride, chloroquine phosphate,metronidazole, metronidazole hydrochloride, pentamidine isethionate. Theat least one anthelmintic can be at least one selected from mebendazole,pyrantel pamoate, thiabendazole. The at least one antifungal can be atleast one selected from amphotericin B, amphotericin B cholesterylsulfate complex, amphotericin B lipid complex, amphotericin B liposomal,fluconazole, flucytosine, griseofulvin microsize, griseofulvinultramicrosize, itraconazole, ketoconazole, nystatin, terbinafinehydrochloride. The at least one antimalarial can be at least oneselected from chloroquine hydrochloride, chloroquine phosphate,doxycycline, hydroxychloroquine sulfate, mefloquine hydrochloride,primaquine phosphate, pyrimethamine, pyrimethamine with sulfadoxine. Theat least one antituberculotic or antileprotic can be at least oneselected from clofazimine, cycloserine, dapsone, ethambutolhydrochloride, isoniazid, pyrazinamide, rifabutin, rifampin,rifapentine, streptomycin sulfate. The at least one aminoglycoside canbe at least one selected from amikacin sulfate, gentamicin sulfate,neomycin sulfate, streptomycin sulfate, tobramycin sulfate. The at leastone penicillin can be at least one selected from amoxcillin/clavulanatepotassium, amoxicillin trihydrate, ampicillin, ampicillin sodium,ampicillin trihydrate, ampicillin sodium/sulbactam sodium, cloxacillinsodium, dicloxacillin sodium, mezlocillin sodium, nafcillin sodium,oxacillin sodium, penicillin G benzathine, penicillin G potassium,penicillin G procaine, penicillin G sodium, penicillin V potassium,piperacillin sodium, piperacillin sodium/tazobactam sodium, ticarcillindisodium, ticarcillin disodium/clavulanate potassium. The at least onecephalosporin can be at least one selected from at least one ofcefaclor, cefadroxil, cefazolin sodium, cefdinir, cefepimehydrochloride, cefixime, cefmetazole sodium, cefonicid sodium,cefoperazone sodium, cefotaxime sodium, cefotetan disodium, cefoxitinsodium, cefpodoxime proxetil, cefprozil, ceftazidime, ceftibuten,ceftizoxime sodium, ceftriaxone sodium, cefuroxime axetil, cefuroximesodium, cephalexin hydrochloride, cephalexin monohydrate, cephradine,loracarbef. The at least one tetracycline can be at least one selectedfrom demeclocycline hydrochloride, doxycycline calcium, doxycyclinehyclate, doxycycline hydrochloride, doxycycline monohydrate, minocyclinehydrochloride, tetracycline hydrochloride. The at least one sulfonamidecan be at least one selected from co-trimoxazole, sulfadiazine,sulfamethoxazole, sulfisoxazole, sulfisoxazole acetyl. The at least onefluoroquinolone can be at least one selected from alatrofloxacinmesylate, ciprofloxacin, enoxacin, levofloxacin, lomefloxacinhydrochloride, nalidixic acid, norfloxacin, ofloxacin, sparfloxacin,trovafloxacin mesylate. The at least one fluoroquinolone can be at leastone selected from alatrofloxacin mesylate, ciprofloxacin, enoxacin,levofloxacin, lomefloxacin hydrochloride, nalidixic acid, norfloxacin,ofloxacin, sparfloxacin, trovafloxacin mesylate. The at least oneantiviral can be at least one selected from abacavir sulfate, acyclovirsodium, amantadine hydrochloride, amprenavir, cidofovir, delavirdinemesylate, didanosine, efavirenz, famciclovir, fomivirsen sodium,foscarnet sodium, ganciclovir, indinavir sulfate, lamivudine,lamivudine/zidovudine, nelfinavir mesylate, nevirapine, oseltamivirphosphate, ribavirin, rimantadine hydrochloride, ritonavir, saquinavir,saquinavir mesylate, stavudine, valacyclovir hydrochloride, zalcitabine,zanamivir, zidovudine.

The at least one macroline anti-infective can be at least one selectedfrom azithromycin, ciarithromycin, dirithromycin, erythromycin base,erythromycin estolate, erythromycin ethylsuccinate, erythromycinlactobionate, erythromycin stearate. The at least one miscellaneousanti-infective can be at least one selected from aztreonam, bacitracin,chloramphenicol sodium sucinate, clindamycin hydrochloride, clindamycinpalmitate hydrochloride, clindamycin phosphate, imipenem and cilastatinsodium, meropenem, nitrofurantoin macrocrystals, nitrofurantoinmicrocrystals, quinupristin/dalfopristin, spectinomycin hydrochloride,trimethoprim, vancomycin hydrochloride. (See, e.g., pp. 24-214 ofNursing 2001 Drug Handbook.)

The at least one inotropic can be at least one selected from amrinonelactate, digoxin, milrinone lactate. The at least one antiarrhythmic canbe at least one selected from adenosine, amiodarone hydrochloride,atropine sulfate, bretylium tosylate, diltiazem hydrochloride,disopyramide, disopyramide phosphate, esmolol hydrochloride, flecainideacetate, ibutilide fumarate, lidocaine hydrochloride, mexiletinehydrochloride, moricizine hydrochloride, phenytoin, phenytoin sodium,procainamide hydrochloride, propafenone hydrochloride, propranololhydrochloride, quinidine bisulfate, quinidine gluconate, quinidinepolygalacturonate, quinidine sulfate, sotalol, tocainide hydrochloride,verapamil hydrochloride. The at least one antianginal can be at leastone selected from amlodipidine besylate, amyl nitrite, bepridilhydrochloride, diltiazem hydrochloride, isosorbide dinitrate, isosorbidemononitrate, nadolol, nicardipine hydrochloride, nifedipine,nitroglycerin, propranolol hydrochloride, verapamil, verapamilhydrochloride. The at least one antihypertensive can be at least oneselected from acebutolol hydrochloride, amlodipine besylate, atenolol,benazepril hydrochloride, betaxolol hydrochloride, bisoprolol fumarate,candesartan cilexetil, captopril, carteolol hydrochloride, carvedilol,clonidine, clonidine hydrochloride, diazoxide, diltiazem hydrochloride,doxazosin mesylate, enalaprilat, enalapril maleate, eprosartan mesylate,felodipine, fenoldopam mesylate, fosinopril sodium, guanabenz acetate,guanadrel sulfate, guanfacine hydrochloride, hydralazine hydrochloride,irbesartan, isradipine, labetalol hydrchloride, lisinopril, losartanpotassium, methyldopa, methyldopate hydrochloride, metoprolol succinate,metoprolol tartrate, minoxidil, moexipril hydrochloride, nadolol,nicardipine hydrochloride, nifedipine, nisoldipine, nitroprussidesodium, penbutolol sulfate, perindopril erbumine, phentolamine mesylate,pindolol, prazosin hydrochloride, propranolol hydrochloride, quinaprilhydrochloride, ramipril, telmisartan, terazosin hydrochloride, timololmaleate, trandolapril, valsartan, verapamil hydrochloride The at leastone antilipemic can be at least one selected from atorvastatin calcium,cerivastatin sodium, cholestyramine, colestipol hydrochloride,fenofibrate (micronized), fluvastatin sodium, gemfibrozil, lovastatin,niacin, pravastatin sodium, simvastatin. The at least one miscellaneousCV drug can be at least one selected from abciximab, aiprostadil,arbutamine hydrochloride, cilostazol, clopidogrel bisulfate,dipyridamole, eptifibatide, midodrine hydrochloride, pentoxifylline,ticlopidine hydrochloride, tirofiban hydrochloride. (See, e.g., pp.215-336 of Nursing 2001 Drug Handbook.)

The at least one nonnarcotic analgesic or antipyretic can be at leastone selected from acetaminophen, aspirin, choline magnesiumtrisalicylate, diflunisal, magnesium salicylate. The at least onenonsteroidal anti-inflammatory drug can be at least one selected fromcelecoxib, diclofenac potassium, diclofenac sodium, etodolac, fenoprofencalcium, flurbiprofen, ibuprofen, indomethacin, indomethacin sodiumtrihydrate, ketoprofen, ketorolac tromethamine, nabumetone, naproxen,naproxen sodium, oxaprozin, piroxicam, rofecoxib, sulindac. The at leastone narcotic or opiod analgesic can be at least one selected fromalfentanil hydrochloride, buprenorphine hydrochloride, butorphanoltartrate, codeine phosphate, codeine sulfate, fentanyl citrate, fentanyltransdermal system, fentanyl transmucosal, hydromorphone hydrochloride,meperidine hydrochloride, methadone hydrochloride, morphinehydrochloride, morphine sulfate, morphine tartrate, nalbuphinehydrochloride, oxycodone hydrochloride, oxycodone pectinate, oxymorphonehydrochloride, pentazocine hydrochloride, pentazocine hydrochloride andnaloxone hydrochloride, pentazocine lactate, propoxyphene hydrochloride,propoxyphene napsylate, remifentanil hydrochloride, sufentanil citrate,tramadol hydrochloride. The at least one sedative-hypnotic can be atleast one selected from chloral hydrate, estazolam, flurazepamhydrochloride, pentobarbital, pentobarbital sodium, phenobarbitalsodium, secobarbital sodium, temazepam, triazolam, zaleplon, zolpidemtartrate. The at least one anticonvulsant can be at least one selectedfrom acetazolamide sodium, carbamazepine, clonazepam, clorazepatedipotassium, diazepam, divalproex sodium, ethosuximde, fosphenytoinsodium, gabapentin, lamotrigine, magnesium sulfate, phenobarbital,phenobarbital sodium, phenytoin, phenytoin sodium, phenytoin sodium(extended), primidone, tiagabine hydrochloride, topiramate, valproatesodium, valproic acid. The at least one antidepressant can be at leastone selected from amitriptyline hydrochloride, amitriptyline pamoate,amoxapine, bupropion hydrochloride, citalopram hydrobromide,clomipramine hydrochloride, desipramine hydrochloride, doxepinhydrochloride, fluoxetine hydrochloride, imipramine hydrochloride,imipramine pamoate, mirtazapine, nefazodone hydrochloride, nortriptylinehydrochloride, paroxetine hydrochloride, phenelzine sulfate, sertralinehydrochloride, tranylcypromine sulfate, trimipramine maleate,venlafaxine hydrochloride. The at least one antianxiety drug can be atleast one selected from alprazolam, buspirone hydrochloride,chlordiazepoxide, chlordiazepoxide hydrochloride, clorazepatedipotassium, diazepam, doxepin hydrochloride, hydroxyzine embonate,hydroxyzine hydrochloride, hydroxyzine pamoate, lorazepam, mephrobamate,midazolam hydrochloride, oxazepam. The at least one antipsychotic drugcan be at least one selected from chlorpromazine hydrochloride,clozapine, fluphenazine decanoate, fluephenazine enanthate, fluphenazinehydrochloride, haloperidol, haloperidol decanoate, haloperidol lactate,loxapine hydrochloride, loxapine succinate, mesoridazine besylate,molindone hydrochloride, olanzapine, perphenazine, pimozide,prochlorperazine, quetiapine fumarate, risperidone, thioridazinehydrochloride, thiothixene, thiothixene hydrochloride, trifluoperazinehydrochloride. The at least one central nervous system stimulant can beat least one selected from amphetamine sulfate, caffeine,dextroamphetamine sulfate, doxapram hydrochloride, methamphetaminehydrochloride, methylphenidate hydrochloride, modafinil, pemoline,phentermine hydrochloride. The at least one antiparkinsonian can be atleast one selected from amantadine hydrochloride, benztropine mesylate,biperiden hydrochloride, biperiden lactate, bromocriptine mesylate,carbidopa-levodopa, entacapone, levodopa, pergolide mesylate,pramipexole dihydrochloride, ropinirole hydrochloride, selegilinehydrochloride, tolcapone, trihexyphenidyl hydrochloride. The at leastone miscellaneous central nervous system drug can be at least oneselected from bupropion hydrochloride, donepezil hydrochloride,droperidol, fluvoxamine maleate, lithium carbonate, lithium citrate,naratriptan hydrochloride, nicotine polacrilex, nicotine transdermalsystem, propofol, rizatriptan benzoate, sibutramine hydrochloridemonohydrate, sumatriptan succinate, tacrine hydrochloride, zolmitriptan.(See, e.g., pp. 337-530 of Nursing 2001 Drug Handbook.)

The at least one cholinergic (e.g., parasymathomimetic) can be at leastone selected from bethanechol chloride, edrophonium chloride,neostigmine bromide, neostigmine methylsulfate, physostigminesalicylate, pyridostigmine bromide. The at least one anticholinergicscan be at least one selected from atropine sulfate, dicyclominehydrochloride, glycopyrrolate, hyoscyamine, hyoscyamine sulfate,propantheline bromide, scopolamine, scopolamine butylbromide,scopolamine hydrobromide. The at least one adrenergics(sympathomimetics) can be at least one selected from dobutaminehydrochloride, dopamine hydrochloride, metaraminol bitartrate,norepinephrine bitartrate, phenylephrine hydrochloride, pseudoephedrinehydrochloride, pseudoephedrine sulfate. The at least one adrenergicblocker (sympatholytic) can be at least one selected fromdihydroergotamine mesylate, ergotamine tartrate, methysergide maleate,propranolol hydrochloride. The at least one skeletal muscle relaxant canbe at least one selected from baclofen, carisoprodol, chlorzoxazone,cyclobenzaprine hydrochloride, dantrolene sodium, methocarbamol,tizanidine hydrochloride. The at least one neuromuscular blockers can beat least one selected from atracurium besylate, cisatracurium besylate,doxacurium chloride, mivacurium chloride, pancuronium bromide,pipecuronium bromide, rapacuronium bromide, rocuronium bromide,succinylcholine chloride, tubocurarine chloride, vecuronium bromide.(See, e.g., pp. 531-84 of Nursing 2001 Drug Handbook.)

The at least one antihistamine can be at least one selected frombrompheniramine maleate, cetirizine hydrochloride, chlorpheniraminemaleate, clemastine fumarate, cyproheptadine hydrochloride,diphenhydramine hydrochloride, fexofenadine hydrochloride, loratadine,promethazine hydrochloride, promethazine theoclate, triprolidinehydrochloride. The at least one bronchodilators can be at least oneselected from albuterol, albuterol sulfate, aminophylline, atropinesulfate, ephedrine sulfate, epinephrine, epinephrine bitartrate,epinephrine hydrochloride, ipratropium bromide, isoproterenol,isoproterenol hydrochloride, isoproterenol sulfate, levalbuterolhydrochloride, metaproterenol sulfate, oxtriphylline, pirbuterolacetate, salmeterol xinafoate, terbutaline sulfate, theophylline. The atleast one expectorants or antitussives can be at least one selected frombenzonatate, codeine phosphate, codeine sulfate, dextramethorphanhydrobromide, diphenhydramine hydrochloride, guaifenesin, hydromorphonehydrochloride. The at least one miscellaneous respiratory drug can be atleast one selected from acetylcysteine, beclomethasone dipropionate,beractant, budesonide, calfactant, cromolyn sodium, dornase alfa,epoprostenol sodium, flunisolide, fluticasone propionate, montelukastsodium, nedocromil sodium, palivizumab, triamcinolone acetonide,zafirlukast, zileuton. (See, e.g., pp. 585-642 of Nursing 2001 DrugHandbook.)

The at least one antacid, adsorbents, or antiflatulents can be at leastone selected from aluminum carbonate, aluminum hydroxide, calciumcarbonate, magaldrate, magnesium hydroxide, magnesium oxide,simethicone, sodium bicarbonate. The at least one digestive enymes orgallstone solubilizers can be at least one selected from pancreatin,pancrelipase, ursodiol. The at least one antidiarrheal can be at leastone selected from attapulgite, bismuth subsalicylate, calciumpolycarbophil, diphenoxylate hydrochloride or atropine sulfate,loperamide, octreotide acetate, opium tincture, opium tincure(camphorated). The at least one laxative can be at least one selectedfrom bisocodyl, calcium polycarbophil, cascara sagrada, cascara sagradaaromatic fluidextract, cascara sagrada fluidextract, castor oil,docusate calcium, docusate sodium, glycerin, lactulose, magnesiumcitrate, magnesium hydroxide, magnesium sulfate, methylcellulose,mineral oil, polyethylene glycol or electrolyte solution, psyllium,senna, sodium phosphates. The at least one antiemetic can be at leastone selected from chlorpromazine hydrochloride, dimenhydrinate,dolasetron mesylate, dronabinol, granisetron hydrochloride, meclizinehydrochloride, metocloproamide hydrochloride, ondansetron hydrochloride,perphenazine, prochlorperazine, prochlorperazine edisylate,prochlorperazine maleate, promethazine hydrochloride, scopolamine,thiethylperazine maleate, trimethobenzamide hydrochloride. The at leastone antiulcer drug can be at least one selected from cimetidine,cimetidine hydrochloride, famotidine, lansoprazole, misoprostol,nizatidine, omeprazole, rabeprozole sodium, rantidine bismuth citrate,ranitidine hydrochloride, sucralfate. (See, e.g., pp. 643-95 of Nursing2001 Drug Handbook.) The at least one coricosteroids can be at least oneselected from betamethasone, betamethasone acetate or betamethasonesodium phosphate, betamethasone sodium phosphate, cortisone acetate,dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate,fludrocortisone acetate, hydrocortisone, hydrocortisone acetate,hydrocortisone cypionate, hydrocortisone sodium phosphate,hydrocortisone sodium succinate, methylprednisolone, methylprednisoloneacetate, methylprednisolone sodium succinate, prednisolone, prednisoloneacetate, prednisolone sodium phosphate, prednisolone tebutate,prednisone, triamcinolone, triamcinolone acetonide, triamcinolonediacetate. The at least one androgen or anabolic steroids can be atleast one selected from danazol, fluoxymesterone, methyltestosterone,nandrolone decanoate, nandrolone phenpropionate, testosterone,testosterone cypionate, testosterone enanthate, testosterone propionate,testosterone transdermal system. The at least one estrogen or progestincan be at least one selected from esterified estrogens, estradiol,estradiol cypionate, estradiol/norethindrone acetate transdermal system,estradiol valerate, estrogens (conjugated), estropipate, ethinylestradiol, ethinyl estradiol and desogestrel, ethinyl estradiol andethynodiol diacetate, ethinyl estradiol and desogestrel, ethinylestradiol and ethynodiol diacetate, ethinyl estradiol andlevonorgestrel, ethinyl estradiol and norethindrone, ethinyl estradioland norethindrone acetate, ethinyl estradiol and norgestimate, ethinylestradiol and norgestrel, ethinyl estradiol and norethindrone andacetate and ferrous fumarate, levonorgestrel, medroxyprogesteroneacetate, mestranol and norethindron, norethindrone, norethindroneacetate, norgestrel, progesterone. The at least one gonadroptropin canbe at least one selected from ganirelix acetate, gonadoreline acetate,histrelin acetate, menotropins. The at least one antidiabetic or glucaoncan be at least one selected from acarbose, chlorpropamide, glimepiride,glipizide, glucagon, glyburide, insulins, metformin hydrochloride,miglitol, pioglitazone hydrochloride, repaglinide, rosiglitazonemaleate, troglitazone. The at least one thyroid hormone can be at leastone selected from levothyroxine sodium, liothyronine sodium, liotrix,thyroid. The at least one thyroid hormone antagonist can be at least oneselected from methimazole, potassium iodide, potassium iodide (saturatedsolution), propylthiouracil, radioactive iodine (sodium iodide ¹³¹I),strong iodine solution. The at least one pituitary hormone can be atleast one selected from corticotropin, cosyntropin, desmophressinacetate, leuprolide acetate, repository corticotropin, somatrem,somatropin, vasopressin. The at least one parathyroid-like drug can beat least one selected from calcifediol, calcitonin (human), calcitonin(salmon), caicitriol, dihydrotachysterol, etidronate disodium. (See,e.g., pp. 696-796 of Nursing 2001 Drug Handbook.)

The at least one diuretic can be at least one selected fromacetazolamide, acetazolamide sodium, amiloride hydrochloride,bumetanide, chlorthalidone, ethacrynate sodium, ethacrynic acid,furosemide, hydrochlorothiazide, indapamide, mannitol, metolazone,spironolactone, torsemide, triamterene, urea. The at least oneelectrolyte or replacement solution can be at least one selected fromcalcium acetate, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, calciumlactate, calcium phosphate (dibasic), calcium phosphate (tribasic),dextran (high-molecular-weight), dextran (low-molecular-weight),hetastarch, magnesium chloride, magnesium sulfate, potassium acetate,potassium bicarbonate, potassium chloride, potassium gluconate, Ringer'sinjection, Ringer's injection (lactated), sodium chloride. The at leastone acidifier or alkalinizer can be at least one selected from sodiumbicarbonate, sodium lactate, tromethamine. (See, e.g., pp. 797-833 ofNursing 2001 Drug. Handbook.)

The at least one hematinic can be at least one selected from ferrousfumarate, ferrous gluconate, ferrous sulfate, ferrous sulfate (dried),iron dextran, iron sorbitol, polysaccharide-iron complex, sodium ferricgluconate complex. The at least one anticoagulant can be at least oneselected from ardeparin sodium, dalteparin sodium, danaparoid sodium,enoxaparin sodium, heparin calcium, heparin sodium, warfarin sodium. Theat least one blood derivative can be at least one selected from albumin5%, albumin 25%, antihemophilic factor, anti-inhibitor coagulantcomplex, antithrombin III (human), factor IX (human), factor IX complex,plasma protein fractions. The at least one thrombolytic enzyme can be atleast one selected from alteplase, anistreplase, reteplase(recombinant), streptokinase, urokinase. (See, e.g., pp. 834-66 ofNursing 2001 Drug Handbook.)

The at least one alkylating drug can be at least one selected frombusulfan, carboplatin, carmustine, chlorambucil, cisplatin,cyclophosphamide, ifosfamide, lomustine, mechlorethamine hydrochloride,melphalan, melphalan hydrochloride, streptozocin, temozolomide,thiotepa. The at least one antimetabolite can be at least one selectedfrom capecitabine, cladribine, cytarabine, floxuridine, fludarabinephosphate, fluorouracil, hydroxyurea, mercaptopurine, methotrexate,methotrexate sodium, thioguanine. The at least one antibioticantineoplastic can be at least one selected from bleomycin sulfate,dactinomycin, daunorubicin citrate liposomal, daunorubicinhydrochloride, doxorubicin hydrochloride, doxorubicin hydrochlorideliposomal, epirubicin hydrochloride, idarubicin hydrochloride,mitomycin, pentostatin, plicamycin, valrubicin. The at least oneantineoplastics that alter hormone balance can be at least one selectedfrom anastrozole, bicalutamide, estramustine phosphate sodium,exemestane, flutamide, goserelin acetate, letrozole, leuprolide acetate,megestrol acetate, nilutamide, tamoxifen citrate, testolactone,toremifene citrate. The at least one miscellaneous antineoplastic can beat least one selected from asparaginase, bacillus Calmette-Guerin (BCG)(live intravesical), dacarbazine, docetaxel, etoposide, etoposidephosphate, gemcitabine hydrochloride, irinotecan hydrochloride,mitotane, mitoxantrone hydrochloride, paclitaxel, pegaspargase, porfimersodium, procarbazine hydrochloride, rituximab, teniposide, topotecanhydrochloride, trastuzumab, tretinoin, vinblastine sulfate, vincristinesulfate, vinorelbine tartrate. (See, e.g., pp. 867-963 of Nursing 2001Drug Handbook.)

The at least one immunosuppressant can be at least one selected fromazathioprine, basiliximab, cyclosporine, daclizumab, lymphocyte immuneglobulin, muromonab-CD3, mycophenolate mofetil, mycophenolate mofetilhydrochloride, sirolimus, tacrolimus. The at least one vaccine or toxoidcan be at least one selected from BCG vaccine, cholera vaccine,diphtheria and tetanus toxoids (adsorbed), diphtheria and tetanustoxoids and acellular pertussis vaccine adsorbed, diphtheria and tetanustoxoids and whole-cell pertussis vaccine, Haemophilius b conjugatevaccines, hepatitis A vaccine (inactivated), hepatisis B vaccine(recombinant), influenza virus vaccine 1999-2000 trivalent types A & B(purified surface antigen), influenza virus vaccine 1999-2000 trivalenttypes A & B (subvirion or purified subvirion), influenza virus vaccine1999-2000 trivalent types A & B (whole virion), Japanese encephalitisvirus vaccine (inactivated), Lyme disease vaccine (recombinant OspA),measles and mumps and rubella virus vaccine (live), measles and mumpsand rubella virus vaccine (live attenuated), measles virus vaccine (liveattenuated), meningococcal polysaccharide vaccine, mumps virus vaccine(live), plague vaccine, pneumococcal vaccine (polyvalent), poliovirusvaccine (inactivated), poliovirus vaccine (live, oral, trivalent),rabies vaccine (adsorbed), rabies vaccine (human diploid cell), rubellaand mumps virus vaccine (live), rubella virus vaccine (live,attenuated), tetanus toxoid (adsorbed), tetanus toxoid (fluid), typhoidvaccine (oral), typhoid vaccine (parenteral), typhoid Vi polysaccharidevaccine, varicella virus vaccine, yellow fever vaccine. The at least oneantitoxin or antivenin can be at least one selected from black widowspider antivenin, Crotalidae antivenom (polyvalent), diphtheriaantitoxin (equine), Micrurus fulvius antivenin). The at least one immuneserum can be at least one selected from cytomegalovirus immune globulin(intraveneous), hepatitis B immune globulin (human), immune globulinintramuscular, immune globulin intravenous, rabies immune globulin(human), respiratory syncytial virus immune globulin intravenous(human), Rh₀(D) immune globulin (human), Rh₀(D) immune globulinintravenous (human), tetanus immune globulin (human), varicella-zosterimmune globulin. The at least one biological response modifiers can beat least one selected from aldesleukin, epoetin alfa, filgrastim,glatiramer acetate for injection, interferon alfacon-1, interferonalfa-2a (recombinant), interferon alfa-2b (recombinant), interferonbeta-1a, interferon beta-1b (recombinant), interferon gamma-1b,levamisole hydrochloride, oprelvekin, sargramostim. (See, e.g., pp.964-1040 of Nursing 2001 Drug Handbook.)

The at least one ophthalmic anti-infectives can be selected formbacitracin, chloramphenicol, ciprofloxacin hydrochloride, erythromycin,gentamicin sulfate, ofloxacin 0.3%, polymyxin B sulfate, sulfacetamidesodium 10%, sulfacetamide sodium 15%, sulfacetamide sodium 30%,tobramycin, vidarabine. The at least one ophthalmic anti-inflammatoriescan be at least one selected from dexamethasone, dexamethasone sodiumphosphate, diclofenac sodium 0.1%, fluorometholone, flurbiprofen sodium,ketorolac tromethamine, prednisolone acetate (suspension) prednisolonesodium phosphate (solution). The at least one miotic can be at least oneselected from acetylocholine chloride, carbachol (intraocular),carbachol (topical), echothiophate iodide, pilocarpine, pilocarpinehydrochloride, pilocarpine nitrate. The at least one mydriatic can be atleast one selected from atropine sulfate, cyclopentolate hydrochloride,epinephrine hydrochloride, epinephryl borate, homatropine hydrobromide,phenylephrine hydrochloride, scopolamine hydrobromide, tropicamide. Theat least one ophthalmic vasoconstrictors can be at least one selectedfrom naphazoline hydrochloride, oxymetazoline hydrochloride,tetrahydrozoline hydrochloride. The at least one miscellaneousophthalmics can be at least one selected from apraclonidinehydrochloride, betaxolol hydrochloride, brimonidine tartrate, carteololhydrochloride, dipivefrin hydrochloride, dorzolamide hydrochloride,emedastine difumarate, fluorescein sodium, ketotifen fumarate,latanoprost, levobunolol hydrochloride, metipranolol hydrochloride,sodium chloride (hypertonic), timolol maleate. The at least one otic canbe at least one selected from boric acid, carbamide peroxide,chloramphenicol, triethanolamine polypeptide oleate-condensate. The atleast one nasal drug can be at least one selected from beclomethasonedipropionate, budesonide, ephedrine sulfate, epinephrine hydrochloride,flunisolide, fluticasone propionate, naphazoline hydrochloride,oxymetazoline hydrochloride, phenylephrine hydrochloride,tetrahydrozoline hydrochloride, triamcinolone acetonide, xylometazolinehydrochloride. (See, e.g., pp. 1041-97 of Nursing 2001 Drug Handbook.)

The at least one local anti-infectives can be at least one selected fromacyclovir, amphotericin B, azelaic acid cream, bacitracin, butoconazolenitrate, clindamycin phosphate, clotrimazole, econazole nitrate,erythromycin, gentamicin sulfate, ketoconazole, mafenide acetate,metronidazole (topical), miconazole nitrate, mupirocin, naftifinehydrochloride, neomycin sulfate, nitrofurazone, nystatin, silversulfadiazine, terbinafine hydrochloride, terconazole, tetracyclinehydrochloride, tiocononazole, tolnaftate. The at least one scabicide orpediculicide can be at least one selected from crotamiton, lindane,permethrin, pyrethrins. The at least one topical corticosteroid can beat least one selected from betamethasone dipropionate, betamethasonevalerate, clobetasol propionate, desonide, desoximetasone,dexamethasone, dexamethasone sodium phosphate, diflorasone diacetate,fluocinolone acetonide, fluocinonide, flurandrenolide, fluticasonepropionate, halcionide, hydrocortisone, hydrocortisone acetate,hydrocortisone butyrate, hydrocorisone valerate, mometasone furoate,triamcinolone acetonide. (See, e.g., pp. 1098-1136 of Nursing 2001 DrugHandbook.)

The at least one vitamin or mineral can be at least one selected fromvitamin A, vitamin B complex, cyanocobalamin, folic acid,hydroxocobalamin, leucovorin calcium, niacin, niacinamide, pyridoxinehydrochloride, riboflavin, thiamine hydrochloride, vitamin C, vitamin D,cholecalciferol, ergocalciferol, vitamin D analogue, doxercalciferol,paricalcitol, vitamin E, vitamin K analogue, phytonadione, sodiumfluoride, sodium fluoride (topical), trace elements, chromium, copper,iodine, manganese, selenium, zinc. The at least one calorics can be atleast one selected from amino acid infusions (crystalline), amino acidinfusions in dextrose, amino acid infusions with electrolytes, aminoacid infusions with electrolytes in dextrose, amino acid infusions forhepatic failure, amino acid infusions for high metabolic stress, aminoacid infusions for renal failure, dextrose, fat emulsions, medium-chaintriglycerides. (See, e.g., pp. 1137-63 of Nursing 2001 Drug Handbook.)

EPO mimetic hinge core mimetibody antibody or polypeptide compositionsof the present invention can further comprise at least one of anysuitable and/or effective amount of a composition or pharmaceuticalcomposition comprising at least one EPO mimetic hinge core mimetibodyprotein or antibody to a cell, tissue, organ, animal or patient in needof such modulation, treatment or therapy, optionally further comprisingat least one selected from at least one TNF antagonist (e.g., but notlimited to a TNF chemical or protein antagonist, TNF monoclonal orpolyclonal antibody or fragment, a soluble TNF receptor (e.g., p55, p70or p85) or fragment, fusion polypeptides thereof, or a small moleculeTNF antagonist, e.g., TNF binding protein I or II (TBP-1 or TBP-II),nerelimonmab, infliximab, enteracept, CDP-571, CDP-870, afelimomab,lenercept, and the like), an antirheumatic (e.g., methotrexate,auranofin, aurothioglucose, azathioprine, etanercept, gold sodiumthiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), amuscle relaxant, a narcotic, a non-steroid inflammatory drug (NSAID), ananalgesic, an anesthetic, a sedative, a local anethetic, a neuromuscularblocker, an antimicrobial (e.g., aminoglycoside, an antifungal, anantiparasitic, an antiviral, a carbapenem, cephalosporin, aflurorquinolone, a macrolide, a penicillin, a sulfonamide, atetracycline, another antimicrobial), an antipsoriatic, acorticosteriod, an anabolic steroid, a diabetes related agent, amineral, a nutritional, a thyroid agent, a vitamin, a calcium relatedhormone, an antidiarrheal, an antitussive, an antiemetic, an antiulcer,a laxative, an anticoagulant, an erythropieitin (e.g., epoetin alpha), afilgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), animmunization, an immunoglobulin, an immunosuppressive (e.g.,basiliximab, cyclosporine, daclizumab), a growth hormone, a hormonereplacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, dornase alpha (Pulmozyme), a cytokine or acytokine antagonist. Non-limiting examples of such cytokines include,but are not limted to, any of IL-1 to IL-23. Suitable dosages are wellknown in the art. See, e.g., Wells et al., eds., PharmacotherapyHandbook, 2^(nd) Edition, Appleton and Lange, Stamford, Conn. (2000);PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition,Tarascon Publishing, Loma Linda, Calif. (2000), each of which referencesare entirely incorporated herein by reference.

Such compositions can also include toxin molecules that are associated,bound, co-formulated or co-administered with at least one antibody orpolypeptide of the present invention. The toxin can optionally act toselectively kill the pathologic cell or tissue. The pathologic cell canbe a cancer or other cell. Such toxins can be, but are not limited to,purified or recombinant toxin or toxin fragment comprising at least onefunctional cytotoxic domain of toxin, e.g., selected from at least oneof ricin, diphtheria toxin, a venom toxin, or a bacterial toxin. Theterm toxin also includes both endotoxins and exotoxins produced by anynaturally occurring, mutant or recombinant bacteria or viruses which maycause any pathological condition in humans and other mammals, includingtoxin shock, which can result in death. Such toxins may include, but arenot limited to, enterotoxigenic E. coli heat-labile enterotoxin (LT),heat-stable enterotoxin (ST), Shigella cytotoxin, Aeromonasenterotoxins, toxic shock syndrome toxin-1 (TSST-1), Staphylococcalenterotoxin A (SEA), B (SEB), or C (SEC), Streptococcal enterotoxins andthe like. Such bacteria include, but are not limited to, strains of aspecies of enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli(e.g., strains of serotype 0157:H7), Staphylococcus species (e.g.,Staphylococcus aureus, Staphylococcus pyogenes), Shigella species (e.g.,Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigellasonnei), Salmonella species (e.g., Salmonella typhi, Salmonellacholera-suis, Salmonella enteritidis), Clostridium species (e.g.,Clostridium perfringens, Clostridium dificile, Clostridium botulinum),Camphlobacter species (e.g., Camphlobacter jejuni, Camphlobacter fetus),Heliobacter species, (e.g., Heliobacter pylori), Aeromonas species (e.g.Aeromonas sobria, Aeromonas hydrophila, Aeromonas caviae), Pleisomonasshigelloides, Yersina enterocolitica, Vibrios species (e.g., Vibrioscholerae, Vibrios parahemolyticus), Klebsiella species, Pseudomonasaeruginosa, and Streptococci. See, e.g., Stein, ed., INTERNAL MEDICINE,3rd ed., pp 1-13, Little, Brown and Co., Boston, (1990); Evans et al.,eds., Bacterial Infections of Humans: Epidemiology and Control, 2d. Ed.,pp 239-254, Plenum Medical Book Co., New York (1991); Mandell et al,Principles and Practice of Infectious Diseases, 3d. Ed., ChurchillLivingstone, New York (1990); Berkow et al, eds., The Merck Manual, 16thedition, Merck and Co., Rahway, N.J., 1992; Wood et al, FEMSMicrobiology Immunology, 76:121-134 (1991); Marrack et al, Science,248:705-711 (1990), the contents of which references are incorporatedentirely herein by reference.

EPO mimetic hinge core mimetibody or specified portion or variantcompositions of the present invention can further comprise at least oneof any suitable auxiliary, such as, but not limited to, diluent, binder,stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvantor the like. Pharmaceutically acceptable auxiliaries are preferred.Non-limiting examples of, and methods of preparing such sterilesolutions are well known in the art, such as, but limited to, Gennaro,Ed., Remington's Pharmaceutical Sciences, 18th Edition, Mack PublishingCo. (Easton, Pa.) 1990. Pharmaceutically acceptable carriers can beroutinely selected that are suitable for the mode of administration,solubility and/or stability of the EPO mimetic hinge core mimetibodycomposition as well known in the art or as described herein.

Pharmaceutical excipients and additives useful in the presentcomposition include but are not limited to proteins, peptides, aminoacids, lipids, and carbohydrates (e.g., sugars, includingmonosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatizedsugars such as alditols, aldonic acids, esterified sugars and the like;and polysaccharides or sugar polymers), which can be present singly orin combination, comprising alone or in combination 1-99.99% by weight orvolume. Exemplary protein excipients include serum albumin such as humanserum albumin (HSA), recombinant human albumin (rHA), gelatin, casein,and the like. Representative amino acid/EPO mimetic hinge coremimetibody or specified portion or variant components, which can alsofunction in a buffering capacity, include alanine, glycine, arginine,betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine,leucine, isoleucine, valine, methionine, phenylalanine, aspartame, andthe like. One preferred amino acid is glycine.

Carbohydrate excipients suitable for use in the invention include, forexample, monosaccharides such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitolsorbitol (glucitol), myoinositol and the like. Preferred carbohydrateexcipients for use in the present invention are mannitol, trehalose, andraffinose.

EPO mimetic hinge core mimetibody compositions can also include a bufferor a pH adjusting agent; typically, the buffer is a salt prepared froman organic acid or base. Representative buffers include organic acidsalts such as salts of citric acid, ascorbic acid, gluconic acid,carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalicacid; Tris, tromethamine hydrochloride, or phosphate buffers. Preferredbuffers for use in the present compositions are organic acid salts suchas citrate.

Additionally, the EPO mimetic hinge core mimetibody or specified portionor variant compositions of the invention can include polymericexcipients/additives such as polyvinylpyrrolidones, ficolls (a polymericsugar), dextrates (e.g., cyclodextrins, such as2-hydroxypropyl-β-cyclodextrin), polyethylene glycols, flavoring agents,antimicrobial agents, sweeteners, antioxidants, antistatic agents,surfactants (e.g., polysorbates such as “TWEEN 20” and “TWEEN 80”),lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol),and chelating agents (e.g., EDTA).

These and additional known pharmaceutical excipients and/or additivessuitable for use in the EPO mimetic hinge core mimetibody compositionsaccording to the invention are known in the art, e.g., as listed in“Remington: The Science & Practice of Pharmacy”, 19^(th) ed., Williams &Williams, (1995), and in the “Physician's Desk Reference”, 52^(nd) ed.,Medical Economics, Montvale, N.J. (1998), the disclosures of which areentirely incorporated herein by reference. Preferrred carrier orexcipient materials are carbohydrates (e.g., saccharides and alditols)and buffers (e.g., citrate) or polymeric agents.

Formulations

As noted above, the invention provides for stable formulations, whichcan preferably include a suitable buffer with saline or a chosen salt,as well as optional preserved solutions and formulations containing apreservative as well as multi-use preserved formulations suitable forpharmaceutical or veterinary use, comprising at least one EPO mimetichinge core mimetibody or specified portion or variant in apharmaceutically acceptable formulation. Preserved formulations containat least one known preservative or optionally selected from the groupconsisting of at least one phenol, m-cresol, p-cresol, o-cresol,chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol,formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate),alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkoniumchloride, benzethonium chloride, sodium dehydroacetate and thimerosal,or mixtures thereof in an aqueous diluent. Any suitable concenirailionor mixture can be used as known in the art, such as 0.001-5%, or anyrange or value therein, such as, but not limited to 0.001, 0.003, 0.005,0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4., 0.5, 0.6, 0.7,0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1,2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5,3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range orvalue therein. Non-limiting examples include, no preservative, 0.1-2%m-cresol (e.g., 0.2, 0.3. 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol(e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g.,0.005, 0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9,1.0%), 0.0005-1.0% alkylparaben(s) (e.g., 0.00075, 0.0009, 0.001, 0.002,0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5,0.75, 0.9, 1.0%), and the like.

As noted above, the invention provides an article of manufacture,comprising packaging material and at least one vial comprising asolution of at least one EPO mimetic hinge core mimetibody or specifiedportion or variant with the prescribed buffers and/or preservatives,optionally in an aqueous diluent, wherein said packaging materialcomprises a label that indicates that such solution can be held over aperiod of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60,66, 72 hours or greater. The invention further comprises an article ofmanufacture, comprising packaging material, a first vial comprisinglyophilized at least one EPO mimetic hinge core mimetibody or specifiedportion or variant, and a second vial comprising an aqueous diluent ofprescribed buffer or preservative, wherein said packaging materialcomprises a label that instructs a patient to reconstitute the at leastone EPO mimetic hinge core mimetibody or specified portion or variant inthe aqueous diluent to form a solution that can be held over a period oftwenty-four hours or greater.

The at least one EPO mimetic hinge core mimetibody or specified portionor variant used in accordance with the present invention can be producedby recombinant means, including from mammalian cell or transgenicpreparations, or can be purified from other biological sources, asdescribed herein or as known in the art.

The range of amounts of at least one EPO mimetic hinge core mimetibodyor specified portion or variant in the product of the present inventionincludes amounts yielding upon reconstitution, if in a wet/dry system,concentrations from about 1.0 μg/ml to about 1000 mg/ml, although lowerand higher concentrations are operable and are dependent on the intendeddelivery vehicle, e.g., solution formulations will differ fromtransdermal patch, pulmonary, transmucosal, or osmotic or micro pumpmethods.

Preferably, the aqueous diluent optionally further comprises apharmaceutically acceptable preservative. Preferred preservativesinclude those selected from the group consisting of phenol, m-cresol,p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl,ethyl, propyl, butyl and the like), benzalkonium chloride, benzethoniumchloride, sodium dehydroacetate and thimerosal, or mixtures thereof. Theconcentration of preservative used in the formulation is a concentrationsufficient to yield an anti-microbial effect. Such concentrations aredependent on the preservative selected and are readily determined by theskilled artisan.

Other excipients, e.g. isotonicity agents, buffers, antioxidants,preservative enhancers, can be optionally and preferably added to thediluent. An isotonicity agent, such as glycerin, is commonly used atknown concentrations. A physiologically tolerated buffer is preferablyadded to provide improved pH control. The formulations can cover a widerange of pHs, such as from about pH 4 to about pH 10, and preferredranges from about pH 5 to about pH 9, and a most preferred range ofabout 6.0 to about 8.0. Preferably the formulations of the presentinvention have pH between about 6.8 and about 7.8. Preferred buffersinclude phosphate buffers, most preferably sodium phosphate,particularly phosphate buffered saline (PBS).

Other additives, such as a pharmaceutically acceptable solubilizers likeTween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40(polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene(20) sorbitan monooleate), Pluronic F68 (polyoxyethylenepolyoxypropylene block copolymers), and PEG (polyethylene glycol) ornon-ionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or188, Pluronic® polyls, other block co-polymers, and chelators such asEDTA and EGTA can optionally be added to the formulations orcompositions to reduce aggregation. These additives are particularlyuseful if a pump or plastic container is used to administer theformulation. The presence of pharmaceutically acceptable surfactantmitigates the propensity for the protein to aggregate.

The formulations of the present invention can be prepared by a processwhich comprises mixing at least one EPO mimetic hinge core mimetibody orspecified portion or variant and a preservative selected from the groupconsisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzylalcohol, alkylparaben, (methyl, ethyl, propyl, butyl and the like),benzalkonium chloride, benzethonium chloride, sodium dehydroacetate andthimerosal or mixtures thereof in an aqueous diluent. Mixing the atleast one EPO mimetic hinge core mimetibody or specified portion orvariant and preservative in an aqueous diluent is carried out usingconventional dissolution and mixing procedures. To prepare a suitableformulation, for example, a measured amount of at least one EPO mimetichinge core mimetibody or specified portion or variant in bufferedsolution is combined with the desired preservative in a bufferedsolution in quantities sufficient to provide the protein andpreservative at the desired concentrations. Variations of this processwould be recognized by one of ordinary skill in the art. For example,the order the components are added, whether additional additives areused, the temperature and pH at which the formulation is prepared, areall factors that may be optimized for the concentration and means ofadministration used.

The claimed formulations can be provided to patients as clear solutionsor as dual vials comprising a vial of lyophilized at least one EPOmimetic hinge core mimetibody or specified portion or variant that isreconstituted with a second vial containing water, a preservative and/orexcipients, preferably a phosphate buffer and/or saline and a chosensalt, in an aqueous diluent. Either a single solution vial or dual vialrequiring reconstitution can be reused multiple times and can sufficefor a single or multiple cycles of patient treatment and thus canprovide a more convenient treatment regimen than currently available.

The present claimed articles of manufacture are useful foradministration over a period of immediately to twenty-four hours orgreater. Accordingly, the presently claimed articles of manufactureoffer significant advantages to the patient. Formulations of theinvention can optionally be safely stored at temperatures of from about2 to about 40° C. and retain the biologically activity of the proteinfor extended periods of time, thus, allowing a package label indicatingthat the solution can be held and/or used over a period of 6, 12, 18,24, 36, 48, 72, or 96 hours or greater. If preserved diluent is used,such label can include use up to at least one of 1-12 months, one-half,one and a half, and/or two years.

The solutions of at least one EPO mimetic hinge core mimetibody orspecified portion or variant in the invention can be prepared by aprocess that comprises mixing at least one EPO mimetic hinge coremimetibody or specified portion or variant in an aqueous diluent. Mixingis carried out using conventional dissolution and mixing procedures. Toprepare a suitable diluent, for example, a measured amount of at leastone EPO mimetic hinge core mimetibody or specified portion or variant inwater or buffer is combined in quantities sufficient to provide theprotein and optionally a preservative or buffer at the desiredconcentrations. Variations of this process would be recognized by one ofordinary skill in the art. For example, the order the components areadded, whether additional additives are used, the temperature and pH atwhich the formulation is prepared, are all factors that may be optimizedfor the concentration and means of administration used.

The claimed products can be provided to patients as clear solutions oras dual vials comprising a vial of lyophilized at least one EPO mimetichinge core mimetibody or specified portion or variant that isreconstituted with a second vial containing the aqueous diluent. Eithera single solution vial or dual vial requiring reconstitution can bereused multiple times and can suffice for a single or multiple cycles ofpatient treatment and thus provides a more convenient treatment regimenthan currently available.

The claimed products can be provided indirectly to patients by providingto pharmacies, clinics, or other such institutions and facilities, clearsolutions or dual vials comprising a vial of lyophilized at least oneEPO mimetic hinge core mimetibody or specified portion or variant thatis reconstituted with a second vial containing the aqueous diluent. Theclear solution in this case can be up to one liter or even larger insize, providing a large reservoir from which smaller portions of the atleast one EPO mimetic hinge core mimetibody or specified portion orvariant solution can be retrieved one or multiple times for transferinto smaller vials and provided by the pharmacy or clinic to theircustomers and/or patients.

Recognized devices comprising these single vial systems include thosepen-injector devices for delivery of a solution such as Humaject®NovoPen®, B-D®Pen, AutoPen®, and OptiPen®. Recognized devices comprisinga dual vial system include those pen-injector systems for reconstitutinga lyophilized drug in a cartridge for delivery of the reconstitutedsolution such as the HumatroPen®.

The products presently claimed include packaging material. The packagingmaterial provides, in addition to the information required by theregulatory agencies, the conditions under which the product can be used.The packaging material of the present invention provides instructions tothe patient to reconstitute the at least one EPO mimetic hinge coremimetibody or specified portion or variant in the aqueous diluent toform a solution and to use the solution over a period of 2-24 hours orgreater for the two vial, wet/dry, product. For the single vial,solution product, the label indicates that such solution can be usedover a period of 2-24 hours or greater. The presently claimed productsare useful for human pharmaceutical product use.

The formulations of the present invention can be prepared by a processthat comprises mixing at least one EPO mimetic hinge core mimetibody orspecified portion or variant and a selected buffer, preferably aphosphate buffer containing saline or a chosen salt. Mixing the at leastone EPO mimetic hinge core mimetibody or specified portion or variantand buffer in an aqueous diluent is carried out using conventionaldissolution and mixing procedures. To prepare a suitable formulation,for example, a measured amount of at least one EPO mimetic hinge coremimetibody or specified portion or variant in water or buffer iscombined with the desired buffering agent in water in quantitiessufficient to provide the protein and buffer at the desiredconcentrations. Variations of this process would be recognized by one ofordinary skill in the art. For example, the order the components areadded, whether additional additives are used, the temperature and pH atwhich the formulation is prepared, are all factors that can be optimizedfor the concentration and means of administration used.

The claimed stable or preserved formulations can be provided to patientsas clear solutions or as dual vials comprising a vial of lyophilized atleast one EPO mimetic hinge core mimetibody or specified portion orvariant that is reconstituted with a second vial containing apreservative or buffer and excipients in an aqueous diluent. Either asingle solution vial or dual vial requiring reconstitution can be reusedmultiple times and can suffice for a single or multiple cycles ofpatient treatment and thus provides a more convenient treatment regimenthan currently available.

At least one EPO mimetic hinge core mimetibody or specified portion orvariant in either the stable or preserved formulations or solutionsdescribed herein, can be administered to a patient in accordance withthe present invention via a variety of delivery methods including SC orIM injection; transdermal, pulmonary, transmucosal, implant, osmoticpump, cartridge, micro pump, or other means appreciated by the skilledartisan, as well-known in the art.

Therapeutic Applications

The present invention for mimetibodies also provides a method formodulating or treating anemia, in a cell, tissue, organ, animal, orpatient including, but not limited to, at least one of any anemia,cancer treatment related anemia, radiotherapy or chemotherapy relatedanemia, viral or bacterial infection treatment related anemia, renalanemia, anemia of prematurity, pediatric and/or adult cancer-associatedanemia, anemia associated with lymphoma, myeloma, multple myeloma,AIDS-associated anemia, concomitant treatment for patients with orwithout autologous blood donation awaiting elective surgery, preoperatveand post operative for surgery, autologous blood donation ortransfusion, perioperative management, cyclic neutropenia or Kostmannsyndrome (congenital agranulocytosis), end-stage renal disease, anemiaassociated with dialysis, chronic renal insufficiency, primaryhemopoietic diseases, such as congenital hypoplastic anemia, thalassemiamajor, or sickle cell disease, vaso-occlusive complications of sicklecell disease. Furman et al., Pediatrics 1992; 90: 716-728, GoldbergScience. 1988;242:1412-1415; Paul et al., Exp Hematol. 1984;12:825-830;Erslev et al., Arch Intern Med. 1968;122:230-235; Ersley et al., AnnClin Lab Sci. 1980;10:250-257; Jacobs et al., Nature. 1985;313:806-810;Lin et al., Proc Natl Acad Sci USA. 1985;82:7580-7584; Law et al., ProcNatl Acad Sci USA. 1986;83:6920-6924; Goldwasser et al., J Biol Chem.1974;249:4202-4206; Eaves et a., Blood. 1978;52:1196-1210; Sawyer etal., Blood. 1989;74:103-109; Winearls et al., Lancet. 1986;2:1175-1178;Eschbach et al., N Engl J Med. 1987;316:73-78; Eschbach et al., AnnIntern Med. 1989;111:992-1000, each reference entirely incoporatedherein by reference.

Mimetibodies of the present invention can also be used for non-renalforms of anemia induced, for example, by chronic infections,inflammatory processes, radiation therapy, and cytostatic drugtreatment, and encouraging results in patients with non-renal anemiahave been reported. See, e.g., Abels R I and Rudnick S A Erythropoietin:evolving clinical applications. Experimental Hematology 19: 842-50(1991); Graber S E and Krantz S B Erythropoietin: biology and clinicaluse. Hematology/Oncol. Clin. North Amer. 3: 369-400 (1989); Jelkman Wand Gross A J (eds) Erythropoietin. Springer, Berlin 1989; Koury M J andBondurant M C The molecular mechanism of erythropoietin action. EuropeanJournal of Biochemistry 210: 649-63 (1992); Krantz S B Erythropoietin.Blood 77: 419-34 (1991); Tabbara I A Erythropoietin. Biology andclinical applications. Archives of Internal Medicine 153: 298-304(1993), each entirely incorporated herein by reference.

The present invention also provides a method for modulating or treatingan anemia or blood cell related condition, in a cell, tissue, organ,animal, or patient, wherein said anemia or blood cell related conditionis associated with at least one including, but not limited to, at leastone of immune related disease, cardiovascular disease, infectious,malignant and/or neurologic disease. Such a method can optionallycomprise administering an effective amount of at least one compositionor pharmaceutical composition comprising at least one EPO mimetic hingecore mimetibody or specified portion or variant to a cell, tissue,organ, animal or patient in need of such modulation, treatment ortherapy.

The present invention also provides a method for modulating or treatingcancer/infecteous disease in a cell, tissue, organ, animal or patient,including, but not limited to, at least one of acute or chronicbacterial infection, acute and chronic parasitic or infectiousprocesses, including bacterial, viral and fungal infections, HIVinfection/HIV neuropathy, meningitis, hepatitis, septic arthritis,peritonitis, pneumonia, epiglottitis, e. coli 0157:h7, hemolytic uremicsyndrome/thrombolytic thrombocytopenic purpura, malaria, denguehemorrhagic fever, leishmaniasis, leprosy, toxic shock syndrome,streptococcal myositis, gas gangrene, mycobacterium tuberculosis,mycobacterium avium intracellulare, pneumocystis carinii pneumonia,pelvic inflammatory disease, orchitis/epidydimitis, legionella, lymedisease, influenza a, epstein-barr virus, vital-associatedhemaphagocytic syndrome, vital encephalitis/aseptic meningitis, and thelike; (ii) leukemia, acute leukemia, acute lymphoblastic leukemia (ALL),B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), chromicmyelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), hairycell leukemia, myelodyplastic syndrome (MDS), a lymphoma, Hodgkin'sdisease, a malignamt lymphoma, non-hodgkin's lymphoma, Burkitt'slymphoma, multiple myeloma, Kaposi's sarcoma, colorectal carcinoma,pancreatic carcinoma, nasopharyngeal carcinoma, malignant histiocytosis,paraneoplastic syndrome/hypercalcemia of malignancy, solid tumors,adenocarcinomas, sarcomas, malignant melanoma, and the like; or (iii)neurodegenerative diseases, multiple sclerosis, migraine headache, AIDSdementia complex, demyelinating diseases, such as multiple sclerosis andacute transverse myelitis; extrapyramidal and cerebellar disorders' suchas lesions of the corticospinal system; disorders of the basal gangliaor cerebellar disorders; hyperkinetic movement disorders such asHuntington's Chorea and senile chorea; drug-induced movement disorders,such as those induced by drugs which block CNS dopamine receptors;hypokinetic movement disorders, such as Parkinson's disease; Progressivesupranucleo Palsy; structural lesions of the cerebellum; spinocerebellardegenerations, such as spinal ataxia, Friedreich's ataxia, cerebellarcortical degenerations, multiple systems degenerations (Mencel,Dejerine-Thomas, Shi-Drager, and Machado-Joseph); systemic disorders(Refsum's disease, abetalipoprotemia, ataxia, telangiectasia, andmitochondrial multi.system disorder); demyelinating core disorders, suchas multiple sclerosis, acute transverse myelitis; and disorders of themotor unit' such as neurogenic muscular atrophies (anterior horn celldegeneration, such as amyotrophic lateral sclerosis, infantile spinalmuscular atrophy and juvenile spinal muscular atrophy); Alzheimer'sdisease; Down's Syndrome in middle age; Diffuse Lewy body disease;Senile Dementia of Lewy body type; Wernicke-Korsakoff syndrome; chronicalcoholism; Creutzfeldt-Jakob disease; Subacute sclerosingpanencephalitis, Hallerrorden-Spatz disease; and Dementia pugilistica,and the like. Such a method can optionally comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one TNF antibody or specified portion or variant toa cell, tissue, organ, animal or patient in need of such modulation,treatment or therapy. See, e.g., the Merck Manual, 16^(th) Edition,Merck & Company, Rahway, N.J. (1992)

Such a method can optionally comprise administering an effective amountof at least one composition or pharmaceutical composition comprising atleast one EPO mimetic hinge core mimetibody or specified portion orvariant to a cell, tissue, organ, animal or patient in need of suchmodulation, treatment or therapy.

The present invention also provides a method for modulating or treatingat least one cardiovascular disease in a cell, tissue, organ, animal, orpatient, including, but not limited to, at least one of cardiac stunsyndrome, myocardial infarction, congestive heart failure, stroke,ischemic stroke, hemorrhage, arteriosclerosis, atherosclerosis, diabeticateriosclerotic disease, hypertension, arterial hypertension,renovascular hypertension, syncope, shock, syphilis of thecardiovascular system, heart failure, cor pulmonale, primary pulmonaryhypertension, cardiac arrhythmias, atrial ectopic beats, atrial flutter,atrial fibrillation (sustained or paroxysmal), chaotic or multifocalatrial tachycardia, regular narrow QRS tachycardia, specific arrythmias,ventricular fibrillation, His bundle arrythmias, atrioventricular block,bundle branch block, myocardial ischemic disorders, coronary arterydisease, angina pectoris, myocardial infarction, cardiomyopathy, dilatedcongestive cardiomyopathy, restrictive cardiomyopathy, valvular heartdiseases, endocarditis, pericardial disease, cardiac tumors, aordic andperipheral aneuryisms, aortic dissection, inflammation of the aorta,occulsion of the abdominal aorta and its branches, peripheral vasculardisorders, occulsive arterial disorders, peripheral atherloscleroticdisease, thromboangitis obliterans, functional peripheral arterialdisorders, Raynaud's phenomenon and disease, acrocyanosis,erythromelalgia, venous diseases, venous thrombosis, varicose veins,arteriovenous fistula, lymphederma, lipedema, unstable angina,reperfusion injury, post pump syndrome, ischemia-reperfusion injury, andthe like. Such a method can optionally comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one EPO mimetic hinge core mimetibody or specifiedportion or variant to a cell, tissue, organ, animal or patient in needof such modulation, treatment or therapy.

Any method of the present invention can comprise administering aneffective amount of a composition or pharmaceutical compositioncomprising at least one EPO mimetic hinge core mimetibody or specifiedportion or variant to a cell, tissue, organ, animal or patient in needof such modulation, treatment or therapy. Such a method can optionallyfurther comprise co-administration or combination therapy for treatingsuch immune diseases, wherein the administering of said at least one EPOmimetic hinge core mimetibody, specified portion or variant thereof,further comprises administering, before concurrently, and/or after, atleast one selected from at least one TNF antagonist (e.g., but notlimited to a TNF antibody or fragment, a soluble TNF receptor orfragment, fusion proteins thereof, or a small molecule TNF antagonist),an antirheumatic, a muscle relaxant, a narcotic, a non-steroidanti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative,a local anethetic, a neuromuscular blocker, an antimicrobial (e.g.,aminoglycoside, an antifungal, an antiparasitic, an antiviral, acarbapenem, cephalosporin, a flurorquinolone, a macrolide, a penicillin,a sulfonamide, a tetracycline, another antimicrobial), an antipsoriatic,a corticosteriod, an anabolic steroid, a diabetes related agent, amineral, a nutritional, a thyroid agent, a vitamin, a calcium relatedhormone, an antidiarrheal, an antitussive, an antiemetic, an antiulcer,a laxative, an anticoagulant, an erythropieitin (e.g., epoetin alpha), afilgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), animmunization, an immunoglobulin, an immunosuppressive (e.g.,basiliximab, cyclosporine, daclizumab), a growth hormone, a hormonereplacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methoxyanthine, a cromolyn,an epinephrine or analog, dornase alpha (Pulmozyme), a cytokine or acytokine antagonist. Suitable dosages are well known in the art. See,e.g., Wells et al., eds., Pharmacotherapy Handbook, 2^(nd) Edition,Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, TarasconPocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, LomaLinda, Calif. (2000), each of which references are entirely incorporatedherein by reference.

Mimetibodies can also be used ex vivo, such as in autologous marrowculture. Briefly, bone marrow is removed from a patient prior tochemotherapy and treated with TPO and/or EPO, optionally in combinationwith mimetibodies, optionally in combination with one or more additionalcytokines. The treated marrow is then returned to the patient afterchemotherapy to speed the recovery of the marrow. In addition, TPO,alone and in combination with EPO mimetibodies and/or EPO, can also beused for the ex vivo expansion of marrow or peripheral blood progenitor(PBPC) cells. Prior to chemotherapy treatment, marrow can be stimulatedwith stem cell factor (SCF) or G-CSF to release early progenitor cellsinto peripheral circulation. These progenitors are optionally collectedand concentrated from peripheral blood and then treated in culture withTPO and mimetibodies, optionally in combination with one or more othercytokines, including but not limited to SCF, G-CSF, IL-3, GM-CSF, IL-6or IL-11, to differentiate and proliferate into high-densitymegakaryocyte cultures, which are optionally then be returned to thepatient following high-dose chemotherapy. Doses of TPO for ex vivotreatment of bone marrow will be in the range of 100 pg/ml to 10 ng/ml,preferably 500 pg/ml to 3 ng/ml. Doses of mimetibodies will beequivalent in activity to EPO which can be used from 0.1 units/ml to 20units/ml, preferably from 0.5 units/ml to 2 units/ml, or any range orvalue therein.

TNF antagonists suitable for compositions, combination therapy,co-administration, devices and/or methods of the present invention(further comprising at least one anti body, specified portion andvariant thereof, of the present invention), include, but are not limitedto, anti-TNF antibodies, ligand-binding fragments thereof, and receptormolecules which bind specifically to TNF; compounds which prevent and/orinhibit TNF synthesis, TNF release or its action on target cells, suchas thalidomide, tenidap, phosphodiesterase inhibitors (e.g,pentoxifylline and rolipram), A2b adenosine receptor agonists and A2badenosine receptor enhancers; compounds which prevent and/or inhibit TNFreceptor signalling, such as mitogen activated protein (MAP) kinaseinhibitors; compounds which block and/or inhibit membrane TNF cleavage,such as metalloproteinase inhibitors; compounds which block and/orinhibit TNF activity, such as angiotensin converting enzyme (ACE)inhibitors (e.g., captopril); and compounds which block and/or inhibitTNF production and/or synthesis, such as MAP kinase inhibitors.

As used herein, a “tumor necrosis factor antibody,” “TNF antibody,”“TNFα antibody,” or fragment and the like decreases, blocks, inhibits,abrogates or interferes with TNFα activity in vitro, in situ and/orpreferably in vivo. For example, a suitable TNF human antibody of thepresent invention can bind TNFα and includes anti-TNF antibodies,antigen-binding fragments thereof, and specified mutants or domainsthereof that bind specifically to TNFα. A suitable TNF antibody orfragment can also decrease block, abrogate, interfere, prevent and/orinhibit TNF RNA, DNA or protein synthesis, TNF release, TNF receptorsignaling, membrane TNF cleavage, TNF activity, TNF production and/orsynthesis.

Chimeric antibody cA2 consists of the antigen binding variable region ofthe high-affinity neutralizing mouse anti-human TNFα IgG1 antibody,designated A2, and the constant regions of a human IgG1, kappaimmunoglobulin. The human IgG1 Fc region improves allogeneic antibodyeffector function, increases the circulating serum half-life anddecreases the immunogenicity of the antibody. The avidity and epitopespecificity of the chimeric antibody cA2 is derived from the variableregion of the murine antibody A2. In a particular embodiment, apreferred source for nucleic acids encoding the variable region of themurine antibody A2 is the A2 hybridoma cell line.

Chimeric A2 (cA2) neutralizes the cytotoxic effect of both natural andrecombinant human TNFα in a dose dependent manner. From binding assaysof chimeric antibody cA2 and recombinant human TNFα, the affinityconstant of chimeric antibody cA2 was calculated to be 1.04×10¹⁰M⁻¹.Preferred methods for determining monoclonal antibody specificity andaffinity by competitive inhibition can be found in Harlow, et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1988; Colligan et al., eds., Current Protocolsin Immunology, Greene Publishing Assoc. and Wiley Interscience, NewYork, (1992-2003); Kozbor et al., Immunol. Today, 4:72-79 (1983);Ausubel et al., eds. Current Protocols in Molecular Biology, WileyInterscience, New York (1987-2003); and Muller, Meth. Enzymol.,92:589-601 (1983), which references are entirely incorporated herein byreference.

In a particular embodiment, murine monoclonal antibody A2 is produced bya cell line designated c134A. Chimeric antibody cA2 is produced by acell line designated c168A.

Additional examples of monoclonal anti-TNF antibodies that can be usedin the present invention are described in the art (see, e.g., U.S. Pat.No. 5,231,024; Möller, A. et al., Cytokine 2(3):162-169 (1990); U.S.application Ser. No. 07/943,852 (filed Sep. 11, 1992); Rathjen et al.,International Publication No. WO 91/02078 (published Feb. 21, 1991);Rubin et al., EPO Patent Publication No. 0 218 868 (published Apr. 22,1987); Yone et al., EPO Patent Publication No. 0 288 088 (Oct. 26,1988); Liang, et al., Biochem. Biophys. Res. Comm. 137:847-854 (1986);Meager, et al., Hybridoma 6:305-311 (1987); Fendly et al., Hybridoma6:359-369 (1987); Bringman, et al., Hybridoma 6:489-507 (1987); andHirai, et al., J. Immunol. Meth. 96:57-62 (1987), which references areentirely incorporated herein by reference).

TNF Receptor Molecules

Preferred TNF receptor molecules useful in the present invention arethose that bind TNFα with high affinity (see, e.g., Feldmann et al.,International Publication No. WO 92/07076 (published Apr. 30, 1992);Schall et al., Cell 61:361-370 (1990); and Loetscher et al., Cell61:351-359 (1990), which references are entirely incorporated herein byreference) and optionally possess low immunogenicity. In particular, the55 kDa (p55 TNF-R) and the 75 kDa (p75 TNF-R) TNF cell surface receptorsare useful in the present invention. Truncated forms of these receptors,comprising the extracellular domains (ECD) of the receptors orfunctional portions thereof (see, e.g., Corcoran et al., Eur. J.Biochem. 223:831-840 (1994)), are also useful in the present invention.Truncated forms of the TNF receptors, comprising the ECD, have beendetected in urine and serum as 30 kDa and 40 kDa TNFα inhibitory bindingproteins (Engelmann, H. et al., J. Biol. Chem. 265:1531-1536 (1990)).TNF receptor multimeric molecules and TNF immunoreceptor fusionmolecules, and derivatives and fragments or portions thereof, areadditional examples of TNF receptor molecules which are useful in themethods and compositions of the present invention. The TNF receptormolecules which can be used in the invention are characterized by theirability to treat patients for extended periods with good to excellentalleviation of symptoms and low toxicity. Low immunogenicity and/or highaffinity, as well as other undefined properties, may contribute to thetherapeutic results achieved.

TNF receptor multimeric molecules useful in the present inventioncomprise all or a functional portion of the ECD of two or more TNFreceptors linked via one or more polypeptide linkers or other nonpeptidelinkers, such as polyethylene glycol (PEG). The multimeric molecules canfurther comprise a signal peptide of a secreted protein to directexpression of the multimeric molecule. These multimeric molecules andmethods for their production have been described in U.S. applicationSer. No.08/437,533 (filed May 9, 1995), the content of which is entirelyincorporated herein by reference.

TNF immunoreceptor fusion molecules useful in the methods andcompositions of the present invention comprise at least one portion ofone or more immunoglobulin molecules and all or a functional portion ofone or more TNF receptors. These immunoreceptor fusion molecules can beassembled as monomers, or hetero- or homo-multimers. The immunoreceptorfusion molecules can also be monovalent or multivalent. An example ofsuch a TNF immunoreceptor fusion molecule is TNF receptor/IgG fusionprotein. TNF immunoreceptor fusion molecules and methods for theirproduction have been described in the art (Lesslauer et al., Eur. J.Immunol. 21:2883-2886 (1991); Ashkenazi et al., Proc. Natl. Acad. Sci.USA 88:10535-10539(1991); Peppel et al., J. Exp. Med.174:1483-1489(1991); Kolls et al., Proc. Natl. Acad. Sci. USA 91:215-219(1994); Butler et al., Cytokine 6(6):616-623 (1994); Baker et al., Eur.J. Immunol. 24:2040-2048 (1994); Beutler et al., U.S. Pat. No.5,447,851;and U.S. application Ser. No.08/442,133 (filed May 16, 1995), each ofwhich references are entirely incorporated herein by reference). Methodsfor producing immunoreceptor fusion molecules can also be found in Caponet al., U.S. Pat. No.5,116,964; Capon et al., U.S. Pat. No.5,225,538;and Capon et al., Nature 337:525-531(1989), which references areentirely incorporated herein by reference.

A functional equivalent, derivative, fragment or region of TNF receptormolecule refers to the portion of the TNF receptor molecule, or theportion of the TNF receptor molecule sequence which encodes TNF receptormolecule, that is of sufficient size and sequences to functionallyresemble TNF receptor molecules that can be used in the presentinvention (e.g., bind TNFα with high affinity and possess lowimmunogenicity). A functional equivalent of TNF receptor molecule alsoincludes modified TNF receptor molecules that functionally resemble TNFreceptor molecules that can be used in the present invention (e.g., bindTNFα with high affinity and possess low immunogenicity). For example, afunctional equivalent of TNF receptor molecule can contain a “SILENT”codon or one or more amino acid substitutions, deletions or additions(e.g., substitution of one acidic amino acid for another acidic aminoacid; or substitution of one codon encoding the same or differenthydrophobic amino acid for another codon encoding a hydrophobic aminoacid). See Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Assoc. and Wiley-Interscience, New York (1987-2003).

Cytokines include, but are not limited to all known cytokines. See,e.g., CopewithCytokines.com. Cytokine antagonists include, but are notlimited to, any antibody, fragment or mimetic, any soluble receptor,fragment or mimetic, any small molecule antagonist, or any combinationthereof.

Any method of the present invention can comprise a method for treating aprotein mediated disorder, comprising administering an effective amountof a composition or pharmaceutical composition comprising at least oneEPO mimetic hinge core mimetibody or specified portion or variant to acell, tissue, organ, animal or patient in need of such modulation,treatment or therapy. Such a method can optionally further compriseco-administration or combination therapy for treating such immunediseases, wherein the administering of said at least one EPO mimetichinge core mimetibody, specified portion or variant thereof, furthercomprises administering, before concurrently, and/or after, at least oneselected from at least one other cytokines such as IL-3, -6 and -11;stem cell factor; G-CSF and GM-CSF.

Typically, treatment of pathologic conditions is effected byadministering an effective amount or dosage of at least one EPO mimetichinge core mimetibody composition that total, on average, a range fromat least about 0.01 to 500 milligrams of at least one EPO mimetic hingecore mimetibody or specified portion or variant/kilogram of patient perdose, and preferably from at least about 0.1 to 100 milligrams EPOmimetic hinge core mimetibody or specified portion or variant/kilogramof patient per single or multiple administration, depending upon thespecific activity of contained in the composition. Alternatively, theeffective serum concentration can comprise 0.1-5000 μg/ml serumconcentration per single or multiple adminstration. Suitable dosages areknown to medical practitioners and will, of course, depend upon theparticular disease state, specific activity of the composition beingadministered, and the particular patient undergoing treatment. In someinstances, to achieve the desired therapeutic amount, it can benecessary to provide for repeated administration, i.e., repeatedindividual administrations of a particular monitored or metered dose,where the individual administrations are repeated until the desireddaily dose or effect is achieved.

Preferred doses can optionally include 0.01, 0.02, 0.03, 0.04, 0.05.0.06, 0.07, 0.08, 009, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, and/or 30 mg/kg/administration, or anyrange, value or fraction thereof, or to achieve a serum concentration of0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9, 2.0, 2.5, 2.9, 3.0, 3.5, 3.9,4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5,8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 20, 12.5, 12.9,13.0, 13.5, 13.9, 14.0, 14.5, 4.9, 5.0, 5.5., 5.9, 6.0, 6.5, 6.9, 7.0,7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9,10, 10.5, 10.9, 11, 11.5, 11.9,12, 12.5, 12.9, 13.0, 13.5, 13.9, 14, 14.5, 15, 15.5, 15.9, 16, 16.5,16.9, 17, 17.5, 17.9, 18, 18.5, 18.9, 19, 19.5, 19.9, 20, 20.5, 20.9,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70,75, 80, 85, 90, 96, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000,1500, 2000, 2500, 3000, 3500, 4000, 4500, and/or 5000 μg/ml serumconcentration per single or multiple administration, or any range, valueor fraction thereof.

Alternatively, the dosage administered can vary depending upon knownfactors, such as the pharmacodynamic characteristics of the particularagent, and its mode and route of administration; age, health, and weightof the recipient; nature and extent of symptoms, kind of concurrenttreatment, frequency of treatment, and the effect desired. Usually adosage of active ingredient can be about 0.1 to 100 milligrams perkilogram of body weight. Ordinarily 0.1 to 50, and preferably 0.1 to 10milligrams per kilogram per administration or in sustained release formis effective to obtain desired results.

As a non-limiting example, treatment of humans or animals can beprovided as a one-time or periodic dosage of at least one EPO mimetichinge core mimetibody or specified portion or variant of the presentinvention 0.01 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, perday, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one ofweek 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19or 20, or any combination thereof, using single, infusion or repeateddoses.

Dosage forms (composition) suitable for internal administrationgenerally contain from about 0.0001 milligram to about 500 milligrams ofactive ingredient per unit or container. In these pharmaceuticalcompositions the active ingredient will ordinarily be present in anamount of about 0.5-95% by weight based on the total weight of thecomposition.

For parenteral administration, the EPO mimetic hinge core mimetibody orspecified portion or variant can be formulated as a solution,suspension, emulsion or lyophilized powder in association, or separatelyprovided, with a pharmaceutically acceptable parenteral vehicle.Examples of such vehicles are water, saline, Ringer's solution, dextrosesolution, and 5% human serum albumin. Liposomes and nonaqueous vehiclessuch as fixed oils may also be used. The vehicle or lyophilized powdermay contain additives that maintain isotonicity (e.g., sodium chloride,mannitol) and chemical stability (e.g., buffers and preservatives). Theformulation is sterilized by known or suitable techniques.

Suitable pharmaceutical carriers are described in the most recentedition of Remington's Pharmaceutical Sciences, A. Osol, a standardreference text in this field.

Therapeutic Administration

Many known and developed modes of can be used according to the presentinvention for administering pharmaceutically effective amounts of atleast one EPO mimetic hinge core mimetibody or specified portion orvariant according to the present invention. While pulmonaryadministration is used in the following description, other modes ofadministration can be used according to the present invention withsuitable results.

An EPO mimetic hinge core mimetibody of the present invention can bedelivered in a carrier, as a solution, emulsion, colloid, or suspension,or as a powder, using any of a variety of devices and methods suitablefor administration by inhalation or other modes described here within orknown in the art.

Parenteral Formulations and Administration

Formulations for parenteral administration can contain as commonexcipients sterile water or saline, polyalkylene glycols such aspolyethylene glycol, oils of vegetable origin, hydrogenated naphthalenesand the like. Aqueous or oily suspensions for injection can be preparedby using an appropriate emulsifier or humidifier and a suspending agent,according to known methods. Agents for injection can be a non-toxic,non-orally administrable diluting agent such as aquous solution or asterile injectable solution or suspension in a solvent. As the usablevehicle or solvent, water, Ringer's solution, isotonic saline, etc. areallowed; as an ordinary solvent, or suspending solvent, sterileinvolatile oil can be used. For these purposes, any kind of involatileoil and fatty acid can be used, including natural or synthetic orsemisynthetic fatty oils or fatty acids; natural or synthetic orsemisynthtetic mono- or di- or tri-glycerides. Parental administrationis known in the art and includes, but is not limited to, conventionalmeans of injections, a gas pressured needle-less injection device asdescribed in U.S. Pat. No. 5,851,198, and a laser perforator device asdescribed in U.S. Pat. No. 5,839,446 entirely incorporated herein byreference.

Alternative Delivery

The invention further relates to the administration of at least one EPOmimetic hinge core mimetibody or specified portion or variant byparenteral, subcutaneous, intramuscular, intravenous, bolus, vaginal,rectal, buccal, sublingual, intranasal, or transdermal means. Protein,EPO mimetic hinge core mimetibody or specified portion or variantcompositions can be prepared for use for parenteral (subcutaneous,intramuscular or intravenous) administration particularly in the form ofliquid solutions or suspensions; for use in vaginal or rectaladministration particularly in semisolid forms such as creams andsuppositories; for buccal, or sublingual administration particularly inthe form of tablets or capsules; or intranasally particularly in theform of powders, nasal drops or aerosols or certain agents; ortransdermally particularly in the form of a gel, ointment, lotion,suspension or patch delivery system with chemical enhancers such asdimethyl sulfoxide to either modify the skin structure or to increasethe drug concentration in the transdermal patch (Junginger, et al. In“Drug Permeation Enhancement”; Hsieh, D. S., Eds., pp. 59-90 (MarcelDekker, Inc. New York 1994, entirely incorporated herein by reference),or with oxidizing agents that enable the application of formulationscontaining proteins and peptides onto the skin (WO 98/53847), orapplications of electric fields to create transient transport pathwayssuch as electroporation, or to increase the mobility of charged drugsthrough the skin such as iontophoresis, or application of ultrasoundsuch as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402) (the abovepublications and patents being entirely incorporated herein byreference).

Pulmonary/Nasal Administration

For pulmonary administration, preferably at least one EPO mimetic hingecore mimetibody or specified portion or variant composition is deliveredin a particle size effective for reaching the lower airways of the lungor sinuses. According to the invention, at least one EPO mimetic hingecore mimetibody or specified portion or variant can be delivered by anyof a variety of inhalation or nasal devices known in the art foradministration of a therapeutic agent by inhalation. These devicescapable of depositing aerosolized formulations in the sinus cavity oralveoli of a patient include metered dose inhalers, nebulizers, drypowder generators, sprayers, and the like. Other devices suitable fordirecting the pulmonary or nasal administration of EPO mimetic hingecore mimetibody or specified portion or variants are also known in theart. All such devices can use of formulations suitable for theadministration for the dispensing of EPO mimetic hinge core mimetibodyor specified portion or variant in an aerosol. Such aerosols can becomprised of either solutions (both aqueous and non aqueous) or solidparticles. Metered dose inhalers like the Ventolin® metered doseinhaler, typically use a propellent gas and require actuation duringinspiration (See, e.g., WO 94/16970, WO 98/35888). Dry powder inhalerslike Turbuhaler® (Astra), Rotahaler® (Glaxo), Diskus® (Glaxo), Spiros™inhaler (Dura), devices marketed by Inhale Therapeutics, and theSpinhaler® powder inhaler (Fisons), use breath-actuation of a mixedpowder (U.S. Pat. No. 4,668,218 Astra, EP 237507 Astra, WO 97/25086Glaxo, WO 94/08552 Dura, U.S. Pat. No. 5,458,135 Inhale, WO 94/06498Fisons, entirely incorporated herein by reference). Nebulizers likeAERx™ Aradigm, the Ultravent® nebulizer (Mallinckrodt), and the AcornII® nebulizer (Marquest Medical Products) (U.S. Pat. No. 5,404,871Aradigm, WO 97/22376), the above references entirely incorporated hereinby reference, produce aerosols from solutions, while metered doseinhalers, dry powder inhalers, etc. generate small particle aerosols.These specific examples of commercially available inhalation devices areintended to be a representative of specific devices suitable for thepractice of this invention, and are not intended as limiting the scopeof the invention. Preferably, a composition comprising at least one EPOmimetic hinge core mimetibody or specified portion or variant isdelivered by a dry powder inhaler or a sprayer. There are a severaldesirable features of an inhalation device for administering at leastone EPO mimetic hinge core mimetibody or specified portion or variant ofthe present invention. For example, delivery by the inhalation device isadvantageously reliable, reproducible, and accurate. The inhalationdevice can optionally deliver small dry particles, e.g. less than about10 μm, preferably about 1-5 μm, for good respirability.

Administration of EPO Mimetic Hinge Core Mimetibody or Specified Portionor Variant Compositions as a Spray

A spray including EPO mimetic hinge core mimetibody or specified portionor variant composition protein can be produced by forcing a suspensionor solution of at least one EPO mimetic hinge core mimetibody orspecified portion or variant through a nozzle under pressure. The nozzlesize and configuration, the applied pressure, and the liquid feed ratecan be chosen to achieve the desired output and particle size. Anelectrospray can be produced, for example, by an electric field inconnection with a capillary or nozzle feed. Advantageously, particles ofat least one EPO mimetic hinge core mimetibody or specified portion orvariant composition protein delivered by a sprayer have a particle sizeless than about 10 μm, preferably in the range of about 1 μm to about 5μm, and most preferably about 2 μm to about 3 μm.

Formulations of at least one EPO mimetic hinge core mimetibody orspecified portion or variant composition protein suitable for use with asprayer typically include EPO mimetic hinge core mimetibody or specifiedportion or variant composition protein in an aqueous solution at aconcentration of about 1 mg to about 20 mg of at least one EPO mimetichinge core mimetibody or specified portion or variant compositionprotein per ml of solution. The formulation can include agents such asan excipient, a buffer, an isotonicity agent, a preservative, asurfactant, and, preferably, zinc. The formulation can also include anexcipient or agent for stabilization of the EPO mimetic hinge coremimetibody or specified portion or variant composition protein, such asa buffer, a reducing agent, a bulk protein, or a carbohydrate. Bulkproteins useful in formulating EPO mimetic hinge core mimetibody orspecified portion or variant composition proteins include albumin,protamine, or the like. Typical carbohydrates useful in formulating EPOmimetic hinge core mimetibody or specified portion or variantcomposition proteins include sucrose, mannitol, lactose, trehalose,glucose, or the like. The EPO mimetic hinge core mimetibody or specifiedportion or variant composition protein formulation can also include asurfactant, which can reduce or prevent surface-induced aggregation ofthe EPO mimetic hinge core mimetibody or specified portion or variantcomposition protein caused by atomization of the solution in forming anaerosol. Various conventional surfactants can be employed, such aspolyoxyethylene fatty acid esters and alcohols, and polyoxyethylenesorbitol fatty acid esters. Amounts will generally range between 0.001and 14% by weight of the formulation. Especially preferred surfactantsfor purposes of this invention are polyoxyethylene sorbitan monooleate,polysorbate 80, polysorbate 20, or the like. Additional agents known inthe art for formulation of a protein such as mimetibodies, or specifiedportions or variants, can also be included in the formulation.

Administration of EPO Mimetic Hinge Core Mimetibody or Specified Portionor Variant Compositions by a Nebulizer

EPO mimetic hinge core mimetibody or specified portion or variantcomposition protein can be administered by a nebulizer, such as jetnebulizer or an ultrasonic nebulizer. Typically, in a jet nebulizer, acompressed air source is used to create a high-velocity air jet throughan orifice. As the gas expands beyond the nozzle, a low-pressure regionis created, which draws a solution of EPO mimetic hinge core mimetibodyor specified portion or variant composition protein through a capillarytube connected to a liquid reservoir. The liquid stream from thecapillary tube is sheared into unstable filaments and droplets as itexits the tube, creating the aerosol. A range of configurations, flowrates, and baffle types can be employed to achieve the desiredperformance characteristics from a given jet nebulizer. In an ultrasonicnebulizer, high-frequency electrical energy is used to createvibrational, mechanical energy, typically employing a piezoelectrictransducer. This energy is transmitted to the formulation of EPO mimetichinge core mimetibody or specified portion or variant compositionprotein either directly or through a coupling fluid, creating an aerosolincluding the EPO mimetic hinge core mimetibody or specified portion orvariant composition protein. Advantageously, particles of EPO mimetichinge core mimetibody or specified portion or variant compositionprotein delivered by a nebulizer have a particle size less than about 10μm, preferably in the range of about 1 μm to about 5 μm, and mostpreferably about 2 μm to about 3 μm.

Formulations of at least one EPO mimetic hinge core mimetibody orspecified portion or variant suitable for use with a nebulizer, eitherjet or ultrasonic, typically include EPO mimetic hinge core mimetibodyor specified portion or variant composition protein in an aqueoussolution at a concentration of about 1 mg to about 20 mg of at least oneEPO mimetic hinge core mimetibody or specified portion or variantprotein per ml of solution. The formulation can include agents such asan excipient, a buffer, an isotonicity agent, a preservative, asurfactant, and, preferably, zinc. The formulation can also include anexcipient or agent for stabilization of the at least one EPO mimetichinge core mimetibody or specified portion or variant compositionprotein, such as a buffer, a reducing agent, a bulk protein, or acarbohydrate. Bulk proteins useful in formulating at least one EPOmimetic hinge core mimetibody or specified portion or variantcomposition proteins include albumin, protamine, or the like. Typicalcarbohydrates useful in formulating at least one EPO mimetic hinge coremimetibody or specified portion or variant include sucrose, mannitol,lactose, trehalose, glucose, or the like. The at least one EPO mimetichinge core mimetibody or specified portion or variant formulation canalso include a surfactant, which can reduce or prevent surface-inducedaggregation of the at least one EPO mimetic hinge core mimetibody orspecified portion or variant caused by atomization of the solution informing an aerosol. Various conventional surfactants can be employed,such as polyoxyethylene fatty acid esters and alcohols, andpolyoxyethylene sorbital fatty acid esters. Amounts will generally rangebetween 0.001 and 4% by weight of the formulation. Especially preferredsurfactants for purposes of this invention are polyoxyethylene sorbitanmono-oleate, polysorbate 80, polysorbate 20, or the like. Additionalagents known in the art for formulation of a protein such as at leastone EPO mimetic hinge core mimetibody or specified portion or variantprotein can also be included in the formulation.

Administration of EPO Mimetic Hinge Core Mimetibody or Specified Portionor Variant Compositions by a Metered Dose Inhaler

In a metered dose inhaler (MDI), a propellant, at least one EPO mimetichinge core mimetibody or specified portion or variant, and anyexcipients or other additives are contained in a canister as a mixtureincluding a liquefied compressed gas. Actuation of the metering valvereleases the mixture as an aerosol, preferably containing particles inthe size range of less than about 10 μm, preferably about 1 μm to about5 μm, and most preferably about 2 μm to about 3 μm. The desired aerosolparticle size can be obtained by employing a formulation of EPO mimetichinge core mimetibody or specified portion or variant compositionprotein produced by various methods known to those of skill in the art,including jet-milling, spray drying, critical point condensation, or thelike. Preferred metered dose inhalers include those manufactured by 3Mor Glaxo and employing a hydrofluorocarbon propellant.

Formulations of at least one EPO mimetic hinge core mimetibody orspecified portion or variant for use with a metered-dose inhaler devicewill generally include a finely divided powder containing at least oneEPO mimetic hinge core mimetibody or specified portion or variant as asuspension in a non-aqueous medium, for example, suspended in apropellant with the aid of a surfactant. The propellant can be anyconventional material employed for this purpose, such aschlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a(hydrofluroalkane-134a), HFA-227 (hydrofluroalkane-227), or the like.Preferably the propellant is a hydrotluorocarbon. The surfactant can bechosen to stabilize the at least one EPO mimetic hinge core mimetibodyor specified portion or variant as a suspension in the propellant, toprotect the active agent against chemical degradation, and the like.Suitable surfactants include sorbitan trioleate, soya lecithin, oleicacid, or the like. In some cases solution aerosols are preferred usingsolvents such as ethanol. Additional agents known in the art forformulation of a protein such as protein can also be included in theformulation.

One of ordinary skill in the art will recognize that the methods of thecurrent invention can be achieved by pulmonary administration of atleast one EPO mimetic hinge core mimetibody or specified portion orvariant compositions via devices not described herein.

Mucosal Formulations and Administration

For absorption through mucosal surfaces, compositions and methods ofadministering at least one EPO mimetic hinge core mimetibody orspecified portion or variant include an emulsion comprising a pluralityof submicron particles, a mucoadhesive macromolecule, a bioactivepeptide, and an aqueous continuous phase, which promotes absorptionthrough mucosal surfaces by achieving mucoadhesion of the emulsionparticles (U.S. Pat. No. 5,514,670). Mucous surfaces suitable forapplication of the emulsions of the present invention can includecorneal, conjunctival, buccal, sublingual, nasal, vaginal, pulmonary,stomachic, intestinal, and rectal routes of administration. Formulationsfor vaginal or rectal administration, e.g. suppositories, can contain asexcipients, for example, polyalkyleneglycols, vaseline, cocoa butter,and the like. Formulations for intranasal administration can be solidand contain as excipients, for example, lactose or can be aqueous oroily solutions of nasal drops. For buccal administration excipientsinclude sugars, calcium stearate, magnesium stearate, pregelinatinedstarch, and the like (U.S. Pat. No. 5,849,695).

Oral Formulations and Administration

Formulations for oral rely on the co-administration of adjuvants (e.g.,resorcinols and nonionic surfactants such as polyoxyethylene oleyl etherand n-hexadecylpolyethylene ether) to increase artificially thepermeability of the intestinal walls, as well as the co-administrationof enzymatic inhibitors (e.g., pancreatic trypsin inhibitors,diisopropylfluorophosphate (DFF) and trasylol) to inhibit enzymaticdegradation. The active constituent compound of the solid-type dosageform for oral administration can be mixed with at least one additive,including sucrose, lactose, cellulose, mannitol, trehalose, raffinose,maltitol, dextran, starches, agar, arginates, chitins, chitosans,pectins, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin,synthetic or semisynthetic polymer, and glyceride. These dosage formscan also contain other type(s) of additives, e.g., inactive dilutingagent, lubricant such as magnesium stearate, paraben, preserving agentsuch as sorbic acid, ascorbic acid, .alpha.-tocopherol, antioxidant suchas cysteine, disintegrator, binder, thickener, buffering agent,sweetening agent, flavoring agent, perfuming agent, etc.

Tablets and pills can be further processed into enteric-coatedpreparations. The liquid preparations for oral administration includeemulsion, syrup, elixir, suspension and solution preparations allowablefor medical use. These preparations may contain inactive diluting agentsordinarily used in said field, e.g., water. Liposomes have also beendescribed as drug delivery systems for insulin and heparin (U.S. Pat.No. 4,239,754). More recently, microspheres of artificial polymers ofmixed amino acids (proteinoids) have been used to deliverpharmaceuticals (U.S. Pat. No. 4,925,673). Furthermore, carriercompounds described in U.S. Pat. No. 5,879,681 and U.S. Pat. No.5,5,871,753 are used to deliver biologically active agents orally areknown in the art.

Transdermal Formulations and Administration

For transdermal administration, the at least one EPO mimetic hinge coremimetibody or specified portion or variant is encapsulated in a deliverydevice such as a liposome or polymeric nanoparticles, microparticle,microcapsule, or microspheres (referred to collectively asmicroparticles unless otherwise stated). A number of suitable devicesare known, including microparticles made of synthetic polymers such aspolyhydroxy acids such as polylactic acid, polyglycolic acid andcopolymers thereof, polyorthoesters, polyanhydrides, andpolyphosphazenes, and natural polymers such as collagen, polyaminoacids, albumin and other proteins, alginate and other polysaccharides,and combinations thereof (U.S. Pat. No. 5,814,599).

Prolonged Administration and Formulations

It can be sometimes desirable to deliver the compounds of the presentinvention to the subject over prolonged periods of time, for example,for periods of one week to one year from a single administration.Various slow release, depot or implant dosage forms can be utilized. Forexample, a dosage form can contain a pharmaceutically acceptablenon-toxic salt of the compounds that has a low degree of solubility inbody fluids, for example, (a) an acid addition salt with a polybasicacid such as phosphoric acid, sulfuric acid, citric acid, tartaric acid,tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenemono- or di-sulfonic acids, polygalacturonic acid, and the like; (b) asalt with a polyvalent metal cation such as zinc, calcium, bismuth,barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and thelike, or with an organic cation formed from e.g.,N,N′-dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of(a) and (b) e.g. a zinc tannate salt. Additionally, the compounds of thepresent invention or, preferably, a relatively insoluble salt such asthose just described, can be formulated in a gel, for example, analuminum monostearate gel with, e.g. sesame oil, suitable for injection.Particularly preferred salts are zinc salts, zinc tannate salts, pamoatesalts, and the like. Another type of slow release depot formulation forinjection would contain the compound or salt dispersed for encapsulatedin a slow degrading, non-toxic, non-antigenic polymer such as apolylactic acid/polyglycolic acid polymer for example as described inU.S. Pat. No. 3,773,919. The compounds or, preferably, relativelyinsoluble salts such as those described above can also be formulated incholesterol matrix silastic pellets, particularly for use in animals.Additional slow release, depot or implant formulations, e.g. gas orliquid liposomes are known in the literature (U.S. Pat. No. 5,770,222and “Sustained and Controlled Release Drug Delivery Systems”, J. R.Robinson ed., Marcel Dekker, Inc., N.Y., 1978).

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

Example 1 Cloning and Expression of an EPO Mimetic Hinge Core Mimetibodyin Mammalian Cells

A typical mammalian expression vector contains at least one promoterelement, which mediates the initiation of transcription of mRNA, the EPOmimetic hinge core mimetibody or specified portion or variant codingsequence, and signals required for the termination of transcription andpolyadenylation of the transcript. Additional elements includeenhancers, Kozak sequences and intervening sequences flanked by donorand acceptor sites for RNA splicing. Highly efficient transcription canbe achieved with the early and late promoters from SV40, the longterminal repeats (LTRS) from Retroviruses, e.g., RSV, HTLVI, HIVI andthe early promoter of the cytomegalovirus (CMV). However, cellularelements can also be used (e.g., the human actin promoter). Suitableexpression vectors for use in practicing the present invention include,for example, vectors such as pIRES1neo, pRetro-Off, pRetro-On, PLXSN, orpLNCX (Clonetech Labs, Palo Alto, Calif.), pcDNA3.1 (±), pcDNA/Zeo (±)or pcDNA3.1/Hygro (±) (Invitrogen), PSVL and PMSG (Pharmacia, Uppsala,Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC67109). Mammalian host cells that could be used include human Hela 293,H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1,quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

Alternatively, the gene can be expressed in stable cell lines thatcontain the gene integrated into a chromosome. The co-transfection witha selectable marker such as dhfr, gpt, neomycin, or hygromycin allowsthe identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded EPO mimetic hinge core mimetibody or specified portion orvariant. The DHFR (dihydrofolate reductase) marker is useful to developcell lines that carry several hundred or even several thousand copies ofthe gene of interest. Another useful selection marker is the enzymeglutamine synthase (GS) (Murphy, et al., Biochem. J. 227:277-279 (1991);Bebbington, et al., Bio/Technology 10:169-175 (1992)). Using thesemarkers, the mammalian cells are grown in selective medium and the cellswith the highest resistance are selected. These cell lines contain theamplified gene(s) integrated into a chromosome. Chinese hamster ovary(CHO) and NSO cells are often used for the production of EPO mimetichinge core mimetibody or specified portion or variants.

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438-447(1985)) plus a fragment of the CMV-enhancer (Boshart, et al., Cell41:521-530 (1985)). Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning ofthe gene of interest. The vectors contain in addition the 3′ intron, thepolyadenylation and termination signal of the rat preproinsulin gene.

Cloning and Expression in CHO Cells

The vector pC4 is used for the expression of EPO mimetic hinge coremimetibody or specified portion or variant. Plasmid pC4 is a derivativeof the plasmid pSV2-dhfr (ATCC Accession No. 37146). The plasmidcontains the mouse DHFR gene under control of the SV40 early promoter.Chinese hamster ovary- or other cells lacking dihydrofolate activitythat are transfected with these plasmids can be selected by growing thecells in a selective medium (e.g., alpha minus MEM, Life Technologies,Gaithersburg, Md.) supplemented with the chemotherapeutic agentmethotrexate. The amplification of the DHFR genes in cells resistant tomethotrexate (MTX) has been well documented (see, e.g., F. W. Alt, etal., J. Biol. Chem. 253:1357-1370 (1978); J. L. Hamlin and C. Ma,Biochem. et Biophys. Acta 1097:107-143 (1990); and M. J. Page and M. A.Sydenham, Biotechnology 9:64-68 (1991)). Cells grown in increasingconcentrations of MTX develop resistance to the drug by overproducingthe target enzyme, DHFR, as a result of amplification of the DHFR gene.If a second gene is linked to the DHFR gene, it is usually co-amplifiedand over-expressed. It is known in the art that this approach can beused to develop cell lines carrying more than 1,000 copies of theamplified gene(s). Subsequently, when the methotrexate is withdrawn,cell lines are obtained that contain the amplified gene integrated intoone or more chromosome(s) of the host cell.

Plasmid pC4 contains for expressing the gene of interest the strongpromoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus(Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985)) plus a fragmentisolated from the enhancer of the immediate early gene of humancytomegalovirus (CMV) (Boshart, et al., Cell 41:521-530(1985)).Downstream of the promoter are BamHI, XbaI, and Asp718 restrictionenzyme cleavage sites that allow integration of the genes. Behind thesecloning sites the plasmid contains the 3′ intron and polyadenylationsite of the rat preproinsulin gene. Other high efficiency promoters canalso be used for the expression, e.g., the human b-actin promoter, theSV40 early or late promoters or the long terminal repeats from otherretroviruses, e.g., HIV and HTLVI. Clontech's Tet-Off and Tet-On geneexpression systems and similar systems can be used to express the EPO ina regulated way in mammalian cells (M. Gossen, and H. Bujard, Proc.Natl. Acad. Sci. USA 89: 5547-5551 (1992)). For the polyadenylation ofthe mRNA other signals, e.g., from the human growth hormone or globingenes can be used as well. Stable cell lines carrying a gene of interestintegrated into the chromosomes can also be selected uponco-transfection with a selectable marker such as gpt, G418 orhygromycin. It is advantageous to use more than one selectable marker inthe beginning, e.g., G418 plus methotrexate.

The plasmid pC4 is digested with restriction enzymes and thendephosphorylated using calf intestinal phosphatase by procedures knownin the art. The vector is then isolated from a 1% agarose gel.

The DNA sequence encoding the complete EPO mimetic hinge core mimetibodyor specified portion or variant is used, corresponding to HC and LCvariable regions of an EPO mimetic hinge core mimetibody of the presentinvention, according to known method steps. Isolated nucleic acidencoding a suitable human constant region (i.e., HC and LC regions) isalso used in this construct.

The isolated variable and constant region encoding DNA and thedephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC4 using,for instance, restriction enzyme analysis.

Chinese hamster ovary (CHO) cells lacking an active DHFR gene are usedfor transfection. 5 μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSV2-neo using lipofectin. The plasmidpSV2neo contains a dominant selectable marker, the neo gene from Tn5encoding an enzyme that confers resistance to a group of antibioticsincluding G418. The cells are seeded in alpha minus MEM supplementedwith 1 μg/ml G418. After 2 days, the cells are trypsinized and seeded inhybridoma cloning plates (Greiner, Germany) in alpha minus MEMsupplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 μg/ml G418.After about 10-14 days single clones are trypsinized and then seeded in6-well petri dishes or 10 ml flasks using different concentrations ofmethotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing atthe highest concentrations of methotrexate are then transferred to new6-well plates containing even higher concentrations of methotrexate (1mM, 2 mM, 5 mM, 10 mM, 20 mM). The same procedure is repeated untilclones are obtained that grow at a concentration of 100-200 mM .Expression of the desired gene product is analyzed, for instance, bySDS-PAGE and Western blot or by reverse phase HPLC analysis.

Example 2 Non-Limiting Example of an EPO mimetic hinge core Mimetibodyof the Invention

Background: EMP-1 (EPO mimetic peptide-1) is a 20 amino acid peptidewith no sequence homology to human erythropoietin (HuEPO), but with theability (as a dimer) to activate the EPO receptor (Wrighton et al, 1996,Science, vol. 273, 458-463). However, its relatively low activity(10,000 to 100,000 fold less than HuEPO) and short half-life (ex-vivohalf-life of 8 hours in 50% serum, in vivo half-life unknown),compromise its utility as a therapeutic. Therefore, a way was needed toconfer upon the peptide a longer half-life, without disturbing, andpossibly improving its potency. To this end, several attempts have beenmade to increase the activity of EMP-1 by stabilizing the dimerizationof the peptide or by incorporating the peptide into larger structures toincrease half-life. Wrighten et al. (1997, Nature Biotechnology, vol.15, 1261-65) combined biotin labeled EMP-1 with streptavidin tostabilize dimerization. They saw a 100 fold increase in activity in anin vitro cell proliferation assay. They also used anti-biotin antibodiesto stabilize the peptide dimer, however only a 10-fold increase inactivity was seen. The same authors prepared a chemically defineddimeric form of EMP-1. In this case an 100-fold increase in activity wasseen in vivo. Another group sought to improve the activity of EMP-1through covalent linkage to polyethylene glycol (PEG) (Johnson et al.,1997, Chem. & Bio., vol. 4(12), 939-50). They reported an increase inpotency of up to 1000 fold, however the construct was found to beimmunogenic in mice (the antibodies were directed to the peptide) (DanaJohnson, Personal communications). Kuai et al. (2000, J. Peptide Res.,vol. 56, 59-62) inserted the EMP-1 peptide into the sequence ofplasminogen activator inhibitor-1, (PAI-1). It was thought that theinsertion of EMP-1 into this scaffold would both stabilize dimerizationand increase half-life. In an in vivo assay the potency of thisconstruct was seen to be significantly higher, such as more than 2500fold higher than EMP-1 alone. It should be noted that different in vitroassays and in vivo models were used in these studies and the reportedpotencies may not be comparable to each other or to results presentedherein.

EPO Mimetic Hinge Core Mimetibody of the Present Invention

A specific, non-limiting, example of this invention is the EMP-hingecore mimetibody construct where V is the first several N-terminal aminoacids of a naturally occurring HC or LC antibody, P is a single copy ofthe bioactive EMP-1 peptide and L is a tandem repeat of either Gly-Seror Gly-Gly-Gly-Ser flexible linker, H is a hinge core region and CH2 &CH3 are of the IgG1 or IgG4 isotype subclass. It is thought that thisstructure will constrain the EMP-1 peptide, but allow sufficientflexibility such that the dimerization of the peptides as part of theassembled homodimer is stabilized. In support of this, the activity ofEMP-hinge core mimetibody in an in vitro cell proliferation assay ismore than 500 fold greater than the EMP-1 peptide and only substantiallysimilar to recombinant HuEPO (rHuEPO). In addition, it is expected thatthe half-life of this construct will be many times that of rHuEPO or theEMP-1 peptide alone and similar to that of an IgG. Consistently, normalmice treated with EMP-hinge core mimetibody attain a significantlyhigher maximal hematocrit compared to mice treated with rHuEPO, whenequal activity units are given, and elevated levels are maintained for alonger period. This construct is efficiently secreted from cells andappears to be properly folded; overcoming problems associated with1^(st) generation mimetibodies.

In addition to the basic structure described above, variants withpotentially favorable biological characteristics are described. Theseinclude constructs that may have a decreased tendency to self-associate,reduced immune effector functions or decreased immunogenicity. Othermodifications that confer desired characteristics such as improvedconformation of the biologically active peptide, and transfer across theblood-brain barrier are envisioned. The proposed variants andmodifications may be combined in any fashion to yield constructs withdesired activities.

Using recombinant DNA methods, the EMP-1 peptide was inserted into anintermediate vector between an immunoglobulin signal peptide and a humanJ sequence. This was done using complementary synthetic oligonucletideswith ends compatible with the restriction sites present in the vectorThese oligonucleotides comprised coding sequences for the signalpeptidase consensus site (QIQ), the EMP-1 peptide (SEQ ID NO:2), and aflexible linker composed of either GS or GGGS. A restriction fragmentcontaining the above-mentioned functional elements was then transferredinto an expression vector. This vector contained the anti-CD4immunoglobulin promoter and enhancer, and the coding sequence for ahuman IgG1 hinge core sequence, and a portion of an IgG1 hinge coreregion, CPPCP (109-113 of SEQ ID NO:66, as shown in FIG. 36C), an HCconstant region 2 (CH2) and constant region 3 (CH3) as well as thenecessary elements for plasmid replication and selection in bacteria andselection for stable expressers in mammalian cells.

This plasmid was linearized and introduced into the NSO mouse myelomacell line via electroporation. Resistant cells were selected and highexpressers of EMP-hinge core mimetibody were identified by ELISA assayof culture supernatants. Purification of the construct from cell culturesupernatants was accomplished by standard proteinA affinitychromatography. Passage of the purified product through SDS-containingpolyacrylamide gels under both denaturing and reducing conditionsconfirmed the expected size of the purified product. The identity of thepurified protein was further confirmed by mass spectrometry andN-terminal sequencing.

The amino acid sequences of EMP-hinge core mimetibodes are shown below.Functional domains are annotated above the peptide coding sequence. Thethree amino acid signal peptide consensus sequence corresponds to thefirst three amino acids of a naturally occurring immunoglobulin. Theseamino acids are thought to contribute to the efficient removal of thesignal peptide by signal peptidase in the endoplasmic reticulum. Thissequence is immediately followed by the EMP-1 coding sequence. The twoC-terminal amino acids of the EMP-1 sequence combined with the next sixamino acids form a flexible linker characterized by the Gly-Gly-Gly-Serrepeat. A human joining (J) region sequence follows. It is thought thatthe J sequence will provide even more flexibility to allow the EMP-1dimmer to assume the proper conformation, and allow the dimmer toprotrude from the globular structure of the immunoglobulin and penetrateinto the cleft between two EPO receptors. The HC hinge region is alsoincluded in the construct immediately following the J region. There arethree cysteines in the IgG1 hinge region (highlighted). The first wouldnormally pair to the immunoglobulin light chain (LC) and the second twoparticipate in interchain bonds between two HCs. The remainder of thesequence is composed of the CH2 & CH3 regions, which constitute the bulkof the protein. One of the reasons that immunoglobulins are believed tohave a long serum half-life is their ability to bind the FcRn thatextends the serum half-life by returning pinocytosed immunoglobulin backto the extracellular space. The binding site of the FcRn overlaps thejunction of the CH2 and CH3 regions (Sheilds et al, 2001, J. Biol.Chem., vol. 276 (9), 6591-6604).

The peptide sequence of EMP-hinge core mimetibody showing importantfunctional domains. V    EMP-1 Peptide     Linker Hinge IgG1 CH2 (SEQ IDNO:82) 1 QIQGGTYSCHFGPLTWVCKPQGG GS    CPPCP APELLGGP IgG1 CH2----------------------------------------------------- 61SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS     ˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜ IgG1CH3 122TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL                              IgG1 CH3 183TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ          IgG1 CH3 241    QGNVFSCSVMHEALHNHYTQKSLSLSPGK V    EMP-1Peptide     Linker Hinge IgG1 CH2 (SEQ ID NO:83) 1QIQGGTYSCHFGPLTWVCKPQGG GGGS  CPPCP APELLGGP IgG1 CH2----------------------------------------------------- 61SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS     ˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜     IgG1 CH3 122TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL                              IgG1 CH3 183TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ             IgG1 CH3 241    QGNVFSCSVNHEALHNHYTQKSLSLSPGK V    EMP-1Peptide     Linker Hinge IgG1 CH2 (SEQ ID NO:84) 1QIQGGTYSCHFGPLTWVCKPQGG GSGGGS CPPCP APELLGGP IgG1 CH2----------------------------------------------------- 61SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS     ˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜     IgG1 CH3 122TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL                              IgG1 CH3 183TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ             IgG1 CH3 241    QGNVFSCSVMHEALHNHYTQKSLSLSPGK V    EMP-1Peptide     Linker Hinge IgG1 CH2 (SEQ ID NO:85) 1QIQGGTYSCHFGPLTWVCKPQGG GS    CPPCP APEAAGGP IgG1 CH2----------------------------------------------------- 61SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS     ˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜     IgG1 CH3 122TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP I EKTI SKAKGQPRE PQVYTLPPSRDEL                              IgG1 CH3 183TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ             IgG1 CH3 241    QGNVFSCSVMHEALHNHYTQKSLSLSPGK V    EMP-1Peptide     Linker Hinge IgG1 CH2 (SEQ ID NO:86) 1QIQGGTYSCHFGPLTWVCKPQGG GGGS    CPPCP APEAAGGP IgG1 CH2----------------------------------------------------- 61SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS     ˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜     IgG1 CH3 122TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL                              IgG1 CH3 183TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ             IgG1 CH3 241    QGNVFSCSVMHEALHNHYTQKSLSLSPGK V    EMP-1Peptide     Linker  Hinge IgG4 CH2 (SEQ ID NO:87) 1QIQGGTYSCHFGPLTWVCKPQGG GS      CPPCP APEFLGGP           IgG 4 CH2---------------------------------------- 61SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS     ˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜     IgG4 CH3 121TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM                                          IgG4 CH3 183TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ                  IgG4 CH3 241         EGNVFSCSVMHEALHNHYTQKSLSLSLGK V   EMP-1 Peptide     Linker  Hinge IgG4 CH2 (SEQ ID NO:88) 1QIQGGTYSCHFGPLTWVCKPQGG GS     CPPCP APEAAGGP           IgG 4 CH2---------------------------------------- 61SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS    ˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜     IgG4 CH3 121TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM                                          IgG4 CH3 183TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ                          IgG4 CH3 241        EGNVFSCSVMHEALHNHYTQKSLSLSLGK V    EMP-1 Peptide     Linker Hinge IgG4 CH2 (SEQ ID NO:89) 1 QIQGGTYSCHFGPLTWVCKPQGG GGGS     CPPCPAPEAAGGP           IgG 4 CH2 ---------------------------------------- 61SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS    ˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜˜     IgG4 CH3 121TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM                                          IgG4 CH3 183TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ                          IgG4 CH3 241        EGNVFSCSVMHEALHNHYTQKSLSLSLGK

It is well known that two IgG heavy chains are assembled during cellularprocessing via disulfide bonds between cysteines located in the hingeregion to form a homodimer. It is expected that this will also occurbetween the modified peptides to form the assembled EMP-hinge coremimetibody construct. In addition, it is expected that the intrachaindisulfide bond between the two cysteines in the EMP-1 peptide will alsoform. The expected structure of EMP-Hinge core mimetibody contains twoEMP-1 peptides. The spatial arrangement of the peptides at theN-terminus along with the flexibility of adjoining sequences shouldallow the peptides to form the bioacive dimer.

The activity of EMP-Hinge core mimetibody was first tested in an invitro bioactivity assay. For this assay, the EPO dependent UT-7/EPO cellline, derived from a patient with acute megakaryoblastic leukemia, wasused (Komatsu et al., 1993, Blood, vol. 82 (2), 456-464). These cellsundergo programmed cell death 48 to 72 hours after withdraw from mediasupplemented with rHuEPO. Cells that have been incubated in the absenceof rHuEPO for 24 hours can be saved if treated with rHuEPO or an EPOagonist. EMP-Hinge core mimetibody was added to cells starved withoutrHuEPO and cell viability was determined 48 hours after treatment usingthe tetrazolium compound MTS (CellTiter 96 Aq_(ueous) One Solution,Promega) that is metabolized by living cells to yield a product with anabsorbance that can be measured. Results of a typical assay showed thepotency of EMP-Hinge core mimetibody on a molar basis to be 500 foldgreater than the EMP-1 peptide and 5 fold less than rHuEPO. In addition,these same cells were stimulated with EMP-Hinge core mimetibody andtyrosine phosphorylation patterns visualized by running cell lysatethrough a polyacrylamide gel. The pattern exhibited by EMP-Hinge coremimetibody was similar to that of rHuEPO, indicating that the mechanismby which EMP-Hinge core mimetibody acts on these cells is like that ofrHuEPO.

In vivo studies were done in normal mice to compare the half-life ofEMP-Hinge core mimetibody to that of rHuEPO and to compare their effectson erythropoiesis. When mice were dosed equally, EMP-Hinge coremimetibody gave a higher maximal response and the response was prolongedcompared to rHuEPO.

The serum concentrations of both rHuEPO and EMP-Hinge core mimetibodywere measured by ELISA. The approximate half-life of EMP hinge coremimetibodies was at least several times that of rHuEPO.

It has been shown that mutation of two lysine (L) residues, L234 & L235,in the IgG1 lower hinge region to alanine (A) will abrogate the abilityof the immunoglobulin to mediate complement dependent cytotoxicity (CDC)and antibody dependant cellular cytotoxicity (ADCC) (Hezereh et al.,2001, J. Virol., vol. 75 (24), 12161-68). Preliminary studies have shownthat EMP-Hinge core mimetibody does not mediate complement lysis ofcells that express the EPO receptor. This may be due to the low numberof receptors that are found on erythroid progenitor cells. In additionthe in vivo expansion of erythriod progenitors as evidenced bysignificant increases in hematocrit supports the possible functionalirrelevance of immune effector functions. However, while no effectorfunction associated affects have been observed, there remains aninterest in introducing these mutations as a precautionary step.

Another modification that would result in a decrease in mediation ofimmune effector functions is the removal of the glycosylation attachmentsite. This can be accomplished by mutation of the asparagine at position297 (N297) to glutamine (Q). Additional changes can optionally includereplacing the threonine (T) with an alternative amino acid to reduce ormodify O-glycosylation, e.g., T34 or T47 with Aglycosylated versions ofthe IgG1 subclass are known to be poor mediators of immune effectorfunction (Jefferis et al. 1998, Immol. Rev., vol. 163, 50-76).

Advantages: The novel construct, EMP-Hinge core mimetibody describedabove offers an alternative way of displaying the bioactive peptideEMP-1. The activity of this construct is in the range of rHuEPO and thein vivo half-life is similar to that of an IgG. In addition, proposedmodifications are expected to, in combination and in addition to thenovel features of EMP-Hinge core mimetibody, enhance the utility of theEMP-Hinge core mimetibody construct.

It will be clear that the invention can be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the present invention

1. At least one EPO mimetic hinge core mimetibody nucleic acid,comprising at least one polynucleotide encoding at least one amino acidsequence of SEQ ID NOS:82 and 84, or a polynucleotide complementarythereto.
 2. At least one EPO mimetic hinge core mimetibody nucleic acid,comprising at least one polynucleotide encoding at least one amino acidsequence of SEQ ID NOS:83 and 85-89, or a polynucleotide complementarythereto.
 3. At least one EPO mimetic hinge core mimetibody nucleic acid,comprising at least one polynucleotide encoding at least one amino acidsequence of SEQ ID NOS:1-30, or a polynucleotide complementary thereto.4. At least one EPO mimetic hinge core mimetibody nucleic acid,comprising at least one polynucleotide encoding a polypeptide accordingto Formula (I):((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is at least one portionof an N-terminus of an immunoglobulin variable region, P is at least onebioactive EPO mimetic peptide, L is a linker sequence, H is at least aportion of an immunoglobulin variable hinge core region, CH2 is at leasta portion of an immunoglobulin CH2 constant region, CH3 is at least aportion of an immunoglobulin CH3 constant region, m, n, o, p, q, r, ands can independently be independently an integer between 0, 1 or 2 and10.
 5. At least one EPO mimetic hinge core mimetibody polypeptide,comprising all of the contiguous amino acids of at least one of SEQ IDNO:82 and
 84. 6. At least one EPO mimetic hinge core mimetibodypolypeptide, comprising all of the contiguous amino acids of at leastone of SEQ ID NO:83 and 85-89.
 7. At least one EPO mimetic hinge coremimetibody polypeptide, comprising all of the contiguous amino acids ofat least one SEQ ID NOS:1-30.
 8. At least one EPO mimetic hinge coremimetibody polypeptide, comprising a polypeptide according to Formula(I):((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is QIQ, P is at leastone bioactive peptide selected from SEQ ID NOS:1-30, L comprises GS,GGGS (SEQ ID NO:73) or GSGGGS (SEQ ID NO:74), H is CPPCP (SEQ ID NO:75),CH2 isAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK(SEQ ID NO:76), CH3 isGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:78), and m, n, o, p, q, r, s are independently an integerbetween 0, 1 or 2 and
 10. 9. At least one EPO mimetic hinge coremimetibody polypeptide, comprising a polypeptide according to Formula(I):((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is QIQ, P is at leastone bioactive peptide selected from SEQ ID NOS:1-30, L comprises GS,GGGS (SEQ ID NO:73) or GSGGGS (SEQ ID NO:74), H is CPPCP (SEQ ID NO:75),CH2 isAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK(SEQ ID NO:77), CH3 isGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:78), and m, n, o, p, q, r, s are independently an integerbetween 0, 1 or 2 and
 10. 10. At least one EPO mimetic hinge coremimetibody polypeptide, comprising a polypeptide according to Formula(I):((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is QIQ, P is at leastone bioactive peptide selected from SEQ ID NOS:1-30, L comprises GS,GGGS (SEQ ID NO:73) or GSGGGS (SEQ ID NO:74), H is CPPCP (SEQ ID NO:75),CH2 isAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK(SEQ ID NO:79), CH3 isGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:81), and m, n, o, p, q, r, s are independently an integerbetween 0, 1 or 2 and
 10. 11. At least one EPO mimetic hinge coremimetibody polypeptide, comprising a polypeptide according to Formula(I):((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is QIQ, P is at leastone bioactive peptide selected from SEQ ID NOS: 1-30, L comprises GS,GGGS (SEQ ID NO:73) or GSGGGS (SEQ ID NO:74), H is CPPCP (SEQ ID NO:75),CH2 isAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSEKTISKAK(SEQ ID NO:80), CH3 isGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:81), and m, n, o, p, q, r, s are independently an integerbetween 0, 1 or 2 and
 10. 12. At least one EPO mimetic hinge coremimetibody polypeptide, comprising a polypeptide according to Formula(I):((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is an N-terminalportion of a human variable region, P is at least one bioactive peptideselected from SEQ ID NOS:1-30, L is linker polypeptide, H is at least aportion of an immunoglobulin variable hinge core region, CH2 isAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK(SEQ ID NO:76), CH3 isGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:78), and m, n, o, p, q, r, s are independently an integerbetween 0, 1 or 2 and
 10. 13. At least one EPO mimetic hinge coremimetibody polypeptide, comprising a polypeptide according to Formula(I):((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is an N-terminalportion of a human variable region, P is at least one bioactive peptideselected from SEQ ID NOS:1-30, L is linker polypeptide, H is at least aportion of an immunoglobulin variable hinge core region, CH2 isAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK(SEQ ID NO:77), CH3 isGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:78), and m, n, o, p, q, r, s are independently an integerbetween 0, 1 or 2 and
 10. 14. At least one EPO mimetic hinge coremimetibody polypeptide, comprising a polypeptide according to Formula(I):((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is an N-terminalportion of a human variable region, P is at least one bioactive peptideselected from SEQ ID NOS:1-30, L is a linker polypeptide, H is at leasta portion of an immunoglobulin variable hinge core region, CH2 isAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSEKTISKAK(SEQ ID NO:79), CH3 isGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:81), and m, n, o, p, q, r, s are independently an integerbetween 0, 1 or 2 and
 10. 15. At least one EPO mimetic hinge coremimetibody polypeptide, comprising a polypeptide according to Formula(I):((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is an N-terminalportion of a human variable region, P is at least one bioactive peptideselected from SEQ ID NOS:1-30, L is a linker polypeptide, H is at leasta portion of an immunoglobulin variable hinge core region, CH2 isAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAK(SEQ ID NO:80), CH3 isGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:81), and m, n, o, p, q, r, s are independently an integerbetween 0, 1 or 2 and
 10. 16. At least one EPO mimetic hinge coremimetibody polypeptide, comprising a polypeptide according to Formula(I):((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is QIQ, P is at leastone bioactive peptide selected from SEQ ID NOS:1-30, L is a linkerpolypeptide, H is at least a portion of an immunoglobulin variable hingecore region, CH2 is at least a portion of an immunoglobulin CH2 constantregion, CH3 is at least a portion of an immunoglobulin CH3 constantregion, and m, n, o, p, q, r, s are independently an integer between 0,1 or 2 and
 10. 17. At least one EPO mimetic hinge core mimetibodypolypeptide, comprising a polypeptide according to Formula (I):((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is at least one portionof an N-terminus of an immunoglobulin variable region, P is at least onebioactive EPO mimetic peptide, L comprises GS, GGGS (SEQ ID NO:73) orGSGGGS (SEQ ID NO:74), H is at least a portion of an immunoglobulinvariable hinge core region, CH2 is at least a portion of animmunoglobulin CH2 constant region, CH3 is at least a portion of animmunoglobulin CH3 constant region, and m, n, o, p, q, r, s areindependently an integer between 0, 1 or 2 and
 10. 18. At least one EPOmimetic hinge core mimetibody polypeptide, comprising a polypeptideaccording to Formula (I):((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is at least one portionof an N-terminus of an immunoglobulin variable region, P is at least onebioactive EPO mimetic peptide, L is a linker polypeptide, H is at leasta portion of an immunoglobulin variable hinge core region, CH2 is atleast a portion of an immunoglobulin CH2 constant region, CH3 is atleast a portion of an immunoglobulin CH3 constant region, and m, n, o,p, q, r, s are independently an integer between 0, 1 or 2 and
 10. 19. Atleast one EPO mimetic hinge core mimetibody polypeptide, comprising apolypeptide according to Formula (I):((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is at least one portionof an N-terminus of an immunoglobulin variable region, P is at least onebioactive EPO mimetic peptide, L is linker polypeptide, H is CPPCP (SEQID NO:75), CH2 is at least a portion of an immunoglobulin CH2 constantregion, CH3 is at least a portion of an immunoglobulin CH3 constantregion, and m, n, o, p, q, r, s are independently an integer between 0,1 or 2 and
 10. 20. At least one EPO mimetic hinge core mimetibodypolypeptide, comprising a polypeptide according to Formula (I):((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is at least one portionof an N-terminus of an immunoglobulin variable region, P is at least onebioactive peptide selected from SEQ ID NOS:1-30, L is a linkerpolypeptide, H is at least a portion of an immunoglobulin variable hingecore region, CH2 is at least a portion of an immunoglobulin CH2 constantregion, CH3 is at least a portion of an immunoglobulin CH3 constantregion, and m, n, o, p, q, r, s are independently an integer between 0,1 or 2 and
 10. 21. An EPO mimetic hinge core mimetibody nucleic acid orEPO mimetic hinge core mimetibody polypeptide according to at least oneof claims 1-20, wherein said polypeptide has at least one activity of atleast one P polypeptide.
 22. An anti-idiotype monoclonal or polyclonalantibody, fusion protein, or fragment thereof, that specifically bindsat least one EPO mimetic hinge core mimetibody polypeptide according toat least one of claims 5-20.
 23. A EPO mimetic hinge core mimetibodynucleic acid encoding at least one EPO mimetic hinge core mimetibodypolypeptide or EPO mimetic hinge core mimetibody antibody according toany of claims 1-20.
 24. A EPO mimetic hinge core mimetibody vectorcomprising at least one isolated nucleic acid according to claim
 23. 25.A EPO mimetic hinge core mimetibody host cell comprising an isolatednucleic acid according to claim
 23. 26. A EPO mimetic hinge coremimetibody host cell according to claim 23, wherein said host cell is atleast one selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2,653, SP2/0, 293, NSO, DG44 CHO, CHO K1, HeLa, myeloma, or lymphomacells, or any derivative, immortalized or transformed cell thereof. 27.A method for producing at least one EPO mimetic hinge core mimetibodypolypeptide or EPO mimetic hinge core mimetibody antibody, comprisingtranslating a nucleic acid according to claim 23 under conditions invitro, in vivo or in situ, such that the EPO mimetic hinge coremimetibody or antibody is expressed in detectable or recoverableamounts.
 28. A composition comprising at least one EPO mimetic hingecore mimetibody nucleic acid, EPO mimetic hinge core mimetibodypolypeptide, or EPO mimetic hinge core mimetibody antibody according toat least one of claims 1-20.
 29. A composition according to claim 28,wherein said composition further comprises at least one pharmaceuticallyacceptable carrier or diluent.
 30. A composition according to claim 28,further comprising at least one composition comprising antherapeutically effective amount of at least one compound, compositionor polypeptide selected from at least one of a detectable label orreporter, a TNF antagonist, an anti-infective drug, a cardiovascular(CV) system drug, a central nervous system (CNS) drug, an autonomicnervous system (ANS) drug, a respiratory tract drug, a gastrointestinal(GI) tract drug, a hormonal drug, a drug for fluid or electrolytebalance, a hematologic drug, an antineoplactic, an immunomodulationdrug, an opthalmic, otic or nasal drug, a topical drug, a nutritionaldrug, a cytokine, or a cytokine antagonist.
 31. A composition accordingto claim 28, in a form of at least one selected from a liquid, gas, ordry, solution, mixture, suspension, emulsion or colloid, a lyophilizedpreparation, or a powder.
 32. A method for diagnosing or treating an EPOligand related condition in a cell, tissue, organ or animal, comprising(a) contacting or administering a composition comprising an effectiveamount of at least one EPO mimetic hinge core mimetibody nucleic acid,polypeptide or antibody according to at least one of claims 1-20, with,or to, said cell, tissue, organ or animal.
 33. A method according toclaim 32, wherein said effective amount is 0.001-50 mg of EPO mimetichinge core mimetibody antibody; 0.000001-500 mg of said EPO mimetichinge core mimetibody; or 0.0001-100 μg of said EPO mimetic hinge coremimetibody nucleic acid per kilogram of said cells, tissue, organ oranimal.
 34. A method according to claim 32, wherein said contacting orsaid administrating is by at least one mode selected from parenteral,subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracelebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,intralesional, bolus, vaginal, rectal, buccal, sublingual, intranasal,or transdermal.
 35. A method according to claim 32, further comprisingadministering, prior, concurrently or after said (a) contacting oradministering, at least one composition comprising an effective amountof at least one compound or polypeptide selected from at least one of adetectable label or reporter, a TNF antagonist, an anti-infective drug,a cardiovascular (CV) system drug, a central nervous system (CNS) drug,an autonomic nervous system (ANS) drug, a respiratory tract drug, agastrointestinal (GI) tract drug, a hormonal drug, a drug for fluid orelectrolyte balance, a hematologic drug, an antineoplactic, animmunomodulation drug, an opthalmic, otic or nasal drug, a topical drug,a nutritional drug, a cytokine, or a cytokine antagonist.
 36. A device,comprising at least one isolated EPO mimetic hinge core mimetibodypolypeptide, antibody or nucleic acid according to at least one ofclaims 1-20, wherein said device is suitable for contacting oradministerting said at least one of said EPO mimetic hinge coremimetibody polypeptide, antibody or nucleic acid, by at least one modeselected from parenteral, subcutaneous, intramuscular, intravenous,intrarticular, intrabronchial, intraabdominal, intracapsular,intracartilaginous, intracavitary, intracelial, intracelebellar,intracerebroventricular, intracolic, intracervical, intragastric,intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, intralesional,bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.37. An article of manufacture for human pharmaceutical or diagnosticuse, comprising packaging material and a container comprising at leastone isolated EPO mimetic hinge core mimetibody polypeptide, antibody ornucleic acid according to at least one of claims 1-20.
 38. The articleof manufacture of claim 32, wherein said container is a component of aparenteral, subcutaneous, intramuscular, intravenous, intrarticular,intrabronchial, intraabdominal, intracapsular, intracartilaginous,intracavitary, intracelial, intracelebellar, intracerebroventricular,intracolic, intracervical, intragastric, intrahepatic, intramyocardial,intraosteal, intrapelvic, intrapericardiac, intraperitoneal,intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal,intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine,intravesical, intralesional, bolus, vaginal, rectal, buccal, sublingual,intranasal, or transdermal delivery device or system.
 39. A method forproducing at least one isolated EPO mimetic hinge core mimetibodypolypeptide, antibody or nucleic acid according to at least one ofclaims 1-20, comprising providing at least one host cell, transgenicanimal, transgenic plant, plant cell capable of expressing in detectableor recoverable amounts said polypeptide, antibody or nucleic acid. 40.At least one EPO mimetic hinge core mimetibody polypeptide, antibody ornucleic acid, produced by a method according to claim 39.